Methods for detecting dna damage and screening for cancer therapeutics

ABSTRACT

A method for detecting DNA damage in a tissue sample involves contacting an immobilized biological sample with a labeled ligand which binds to human 53Bp1, and examining the immobilized sample for the presence of a label generated-detectable signal concentrated in foci in said sample. The presence of concentrated foci is indicative of DNA damage and the presence of diffuse signal is indicative of a normal sample. Diagnostic reagents contain a ligand that binds to human 53Bp1 associated with a detectable label. Diagnostic kits for detecting DNA damage in a biological sample contain such diagnostic reagents and signal detection components. Compositions that inhibit or antagonize the biological activity of 53Bp1 are identified by suitable assays, and are employed in methods of retarding the growth of a cancer cell.

[0001] This invention was supported, at least in part, by National Institute of Health, Grant No. 5RO1 CA76367-03. The United States government has certain rights in this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and compositions for diagnosis of cancer and other consequences of DNA damage in mammalian cells and tissues, and to methods of drug screening for anti-cancer compounds.

BACKGROUND OF THE INVENTION

[0003] The stability or integrity of the genomes of eukaryotes is the result of a complex interplay of functions at the center of which is regulation of DNA damage checkpoints and DNA repair [Petrini, J. H., 1999 Amer. J. Hum. Genet., 64:1264-1269]. In eukaryotes, when the DNA is damaged, the cell must first sense that damage is present, then induce cell cycle arrest by activating an evolutionarily conserved DNA damage checkpoint. The checkpoint causes arrest of the cell cycle at the G1/S and G2/M boundaries and activation of DNA repair functions [Elledge, S. J., 1996 Science, 274:1664-1672; Longhese, M. P. et al, 1998 EMBO J., 17:5525-5528; Weinert, T. 1998 Curr. Opin. Genet. Dev., 8:185-193].

[0004] Different agents cause different types of DNA damage. Genomic instability, which is a hallmark of neoplastic transformation, may result from defects in the cell cycle checkpoint proteins or DNA repair proteins [Hartwell, L. 1992 Cell 71:543-546; Lengauer, C. et al., 1998 Nature, 396:643-649; and Loeb, L. A., 1991 Cancer Res., 51:3075-3079]. One of the most serious threats to genetic integrity is DNA double-strand breaks (DNA DSBs) which are produced from exogenous agents, such as ionizing radiation, and from errors occurring during normal replication or recombination. DNA DSBs are the most important agents of DNA damage from the cell's perspective, because they are the most difficult to repair.

[0005] Considerable information about DNA repair genes and DNA damage checkpoint genes is available. For example, checkpoint proteins are highly conserved and homologues of most are present in S. pombe and higher eukaryotes. Human homologues of S. cerevisiae RAD24, RAD17, MEC3 and DDC1 have been cloned and partially characterized [Bessho, T., and Sancar, A. 2000 J. Biol. Chem., 275:7451-7454; Lieberman, H. B. et al, 1996 Proc. Natl. Acad. Sci. USA, 93:13890-13895; St. Onge, R. P. M. et al, 1999 Mol. Biol. Cell, 10:1985-1995; Volkmer, E., and Karnitz, L. M. 1999 J. Biol. Chem., 274: 567-570]. There are two putative human homologues of Mec1, ATM (ataxia-telangiectasia mutated) [Savitsky, K. et al, 1995 Science, 268:1749-1753] and ATR (AT and rad-related) [Bentley, N. J. et al, 1996 EMBO J., 15:6641-665 1]. In humans, ATM responds to DNA double stranded breaks (DSBs) and when inactivated in patients with ataxia telangiectasia leads to checkpoint defects in G1, S, and G2 [Halazonetis, T. D., and Shiloh, Y. 1999 Biochim Biophys Acta, 1424:R45-55]. Human ATR may mediate the response to DNA damage other than DSBs [Bentley (1996) cited above; Cimprich, K. A. et al, 1996 Proc. Natl. Acad. Sci. USA, 93:2850-2855]. Chk2, the human homologue of S. cerevisiae Rad53, becomes phosphorylated in response to DNA DSBs in an ATM-dependent manner [Blasina, A. et al, 1999 Curr. Biol., 9:1-10; Brown, A. L. et al, 1999 Proc. Natl. Acad. Sci. USA, 96:3745-3750; Matsuoka, S. et al, 1998 Science, 282:1893-1897] leading to stabilization of the tumor suppressor protein p53 and cell cycle arrest in G1 [Chehab, N. H. et al, 2000 Genes Dev., 14:278-288; Matsuoka (1998) cited above]. Germ line mutations in Chk2 are found in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype typically associated with mutations in p53, suggesting that Chk2 is a tumor suppressor gene and when mutated leads to a predisposition to sarcoma, breast cancer, and brain [Bell, D. W. et al, 1999 Science, 286:2528-2531].

[0006] One of the few human homologues that remain to be found is that of budding yeast S. cerevisiae Rad9, which was the first checkpoint protein to be identified [Weinert, T. A., and Hartwell, L. H. 1988 Science, 241:317-322]. Rad9 is a component of the DNA damage checkpoint and is required for cell cycle arrest following genomic insult. Rad9 has two carboxy terminal BRCT (3RCA1 C terminus) domains which are found in many proteins with functions related to the DNA damage response, such as, BRCA1, NBS, XRCC4, DNA ligase 4, PARP, and many others [Bork, P. et al, 1997 Faseb J., 11:68-76; Callebaut, I., and Mornon, J. P. 1997 FEBS _(—) Lett, 400:25-30]. Rad9, along with proteins encoded by genes in the RAD24 epistasis group, including RAD17, RAD24, MEC3, and DDC1 [Longhese (1998), cited above; Paulovich, A. G. et al, 1997 Cell, 88:315-321; Weinert (1998) cited above] are proposed to sense DNA damage and regulate activation and phosphorylation of Mec1, a protein kinase required for subsequent phosphorylation and activation of Rad53/Spk1 and Chk1 kinases. Rad53/Spk1 and Chk1 then phosphorylate proteins that regulate progression through the cell cycle [Sanchez, Y. et al, 1996 Science, 271:357-360; and Sun, Z. et al, 1996 Genes Dev., 10: 395-406].

[0007] However, very little is known about the proteins that actually sense DNA damage. The sensing protein varies depending on the type of DNA damage. For example, different proteins are required for activating the DNA damage checkpoint when the cell is exposed to UV light (which induces pyrimidine dimers) than the proteins that are required to activate the DNA damage checkpoint when the cell is exposed to ionizing radiation (which induces DNA strand breaks). Part of the difficulty in identifying sensor proteins is the inability to observe and/or isolate sites of DNA damage, such as DNA DSBs.

[0008] Proteins that localize to sites of DNA damage are involved in DNA repair and/or checkpoint control. Thus, one approach useful for visualizing DSBs is by immunofluorescence using antibodies to proteins known to localize to such sites. Such an approach has been employed with the Mre11/Rad50/NBS protein complex, which is involved in DNA repair and checkpoint functions. The Mre11/Rad50/NBS complex forms nuclear foci in response to ionizing radiation that localize to sites of DNA DSBs between four and eight hours after irradiation. Approximately 50% of cells contain on average 12 Mre11 foci per cell 8 hours following 12 Gy gamma irradiation [Maser, R. S. et al, 1997 Mol. Cell. Biol., 17: 6087-6096]. Petrini (1999), cited above exposed partially shielded cells to synchrotron generated ultrasoft x-rays followed by immunofluorescence to probe for Mre11. Mre11 relocalized to the non-shielded areas in a striped pattern corresponding to the regions exposed to X-rays [Nelms, B. E. et al., 1998 Science, 280:590-592]. However, DNA breaks occur immediately after X-rays or gamma irradiation and most of them are repaired well before the four hour time point in which Mre11 foci are evident [Lobrich, M. et al, 1995 Proc. Natl. Acad. Sci. USA, 92:12050-12054]. Also, limited accessibility to a synchrotron irradiator does not allow this approach for visualizing DSB's to be used routinely.

[0009] Other proteins that localize to points of DNA damage include BRCA1 [Scully, R. et al, 1997 Cell, 90: 425-435] and Chk2 [Lee, J. S. et al, 2000 Nature, 404:201-204]. BRCA1 and Chk2 form foci predominately in S-phase cells in the absence of DNA damage. These foci disperse within one hour of gamma irradiation and reform approximately 8 hours later [Lee (2000), cited above]. Approximately 10% of cells contain BRCA1 foci that colocalize with the Mre11/Rad50/NBS complex at sites of DNA DSBs [Wang, Y. et al, 2000 Genes Dev., 14:927-939; Zhong, Q. et al, 1999 Science, 285:747-750].

[0010] Still other proteins that have been reported to form nuclear foci or redistribute in the nucleus in response to DNA damage are Rad51 [Haaf, T. et al, 1995 Proc. Natl. Acad. Sci. USA, 92:2298-2302] and Rad54 [Tan, T. L. et al, 1999 Curr. Biol., 9:325-328]. Rad51 and Rad54 form nuclear foci in response to ionizing radiation. The foci increase in number with time following treatment with irradiation [Morrison, C. et al, 2000 EMBO J, 19:463-471 and 19(4):786]. Yet another such protein is BLM, which localizes to punctate nuclear structures normally [Gharibyan, V., and Youssoufian, H. 1999 Mol. Carcinog., 26:261-273].

[0011] Still other proteins known to localize to sites of DNA DSBs, such as the DNA-PK/Ku complex, AIM and ATR, DNA ligase 4, XRCC4 and PARP, do not form visible nuclear foci in response to DNA damage using immunofluorescence [Lindahl, T., and Wood, R. D. 1999 Science, 286:1897-1905].

[0012] Thus, there is at present no method which can use these proteins for the observation and/or isolation of DNA DSBs.

[0013] The human p53-binding protein, 53Bp1 was identified in a yeast two-hybrid assay as a protein that binds the p53 tumor suppressor protein. The 53Bp1 was found to bind to the central DNA binding domain of wildtype, but not mutant, p53 and to enhance p53-mediated transcriptional activation of p21. 53Bp1 was proposed to have a role as a transactivator of p53 [Iwabuchi, K. et al, 1994 Proc. Natl. Acad. Sci. USA, 91:6098-6102; Iwabuchi, K. et al, 1998 J. Biol. Chem., 273:26061-26068]. Although 53Bp1 shares no overall homology to other known proteins, the carboxy terminus contains two tandem BRCT domains which are sufficient for binding to p53 [Iwabuchi (1998), cited above]. The nucleotide and protein sequences of 53Bp1 are provided in GenBank, Accession No. AF078776, submitted Jul. 16, 1998.

[0014] There remains a need in the art for methods and compositions for identifying cells and tissues which have sites of DNA damage, e.g., tumor cells, for diagnostic purposes as well as for screening methods for the identification of useful cancer therapeutics.

SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides a method for detecting DNA damage in a tissue sample. This method involves contacting a biological sample with a ligand which binds to human 53Bp1. The ligand is associated with a label which provides a detectable signal. When the sample, preferably immobilized, is examined for the presence of signal, the signal is either in concentrated foci of 53Bp1 in the sample or diffused throughout the sample. The presence of concentrated foci is indicative of DNA damage and the presence of diffuse signal is indicative of a normal sample.

[0016] In another aspect, the invention provides a diagnostic reagent comprising a ligand that binds to human 53Bp1, the ligand associated with a detectable label.

[0017] In still a further aspect, the invention provides a diagnostic kit for detecting DNA damage in a biological sample. The kit comprises a diagnostic reagent which is a ligand which binds to human 53Bp1, the ligand associated with a detectable label, and suitable components for detection of the label.

[0018] In another aspect, the invention provides a method of screening test compounds to identify a composition that inhibits or antagonizes the biological activity of 53Bp1, such as a small chemical compound, or inhibits the expression of 53Bp1, such as an antisense sequence. The method comprises employing a 53Bp1 ligand associated with a detectable label to detect the expression of 53Bp1 in a cell contacted with a test compound or to detect the presence or number of 53Bp1 induced nuclear foci in cells contacted with a test compound.

[0019] In yet another aspect of the invention, a composition which antagonizes or inhibits the biological activity or expression of 53Bp1 is provided, which composition is optionally identified by the above method.

[0020] In still another aspect of the invention, there is provided a method of retarding the growth of a cancer cell, the method comprising administering to the site of a cancer cell a 53Bp1 inhibitor that prevents the 53Bp1 from performing its DNA repair function or inhibits the expression of 53Bp1.

[0021] In yet a further aspect of the invention, there is provided a method of targeting a tumor cell for delivery of a therapeutic agent, comprising administering to a patient bearing a tumor containing 53Bp1 foci a ligand that binds to 53Bp1, said ligand associated with a compound that retards the growth of, or kills, the tumor cell.

[0022] Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1A is a graph demonstrating the mean number and standard deviation of 53Bp1 foci per cell one hour after exposure to ionizing radiation as a function of the dose of radiation exposure in Gy.

[0024]FIG. 1B is a graph showing the mean number and standard deviation of 53Bp1 foci per cell in cells exposed to 1 Gy ionizing radiation as a function of time in minutes after radiation exposure.

[0025]FIG. 1C is a graph showing the percentage of cells exhibiting 53Bp1 foci within a population of cells exposed to 1 Gy ionizing radiation as a function of time in minutes after radiation exposure.

[0026]FIG. 2A is a graph demonstrating the effect of wortmannin on the kinetics of 53Bp1 foci/cell appearance and disappearance (i.e., the mean number and standard deviation) in cells exposed to 1 Gy ionizing radiation as a function of time in minutes after radiation exposure. Cells exposed to wortmannin (Wort, ); control cells (Ctrl, ∘).

[0027]FIG. 2B is a graph demonstrating the effect of wortmannin on the percent of cells exhibiting 53Bp1 foci within a population of cells exposed to 1 Gy ionizing radiation as a function of time in minutes after radiation exposure. The symbols are identical to those of FIG. 2A.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides novel methods for detecting DNA damage in a cell or tissue, as well as novel diagnostic reagents, and methods and compositions for screening for anti-cancer drugs and treating cancer, based on the human p53-binding protein, hereinafter referred to as 53Bp1.

[0029] A. Characteristics of 53Bp1

[0030] Using the genomes of C. elegans and Drosophila, the inventors characterized 53Bp1 as having a function quite distinct from the function that has been identified for 53Bp1 by the prior art, i.e., as a transactivator of p53. The inventors have identified that 53Bp1 is a DNA damage responsive protein that functions upstream of ATM and is a homologue of budding yeast Rad9 based on the degree of protein similarity between these two proteins. 53Bp1 contains BRCT domains at amino acid residues 1713 to 1973 of SEQ ID NO: 2. Although proteins containing BRCT domains have diverse functions, their common involvement in the cellular response to DNA damage and the link between 53Bp1 and p53 suggest a potential similar role for 53Bp1. The inventors have determined, as evidenced in the examples below, that 53Bp1 functions early in the DNA repair pathway. 53Bp1 localizes early to DNA DSBs in a time and dose-dependent manner which is ATM-independent and then colocalizes with the Mre11/Rad50/NBS-p95 complex. These data indicate that 53Bp1 participates in the cellular responses to DNA damage and functions as a DNA checkpoint or repair protein and, therefore, participate in the maintenance of genome integrity. This protein localizes to sites of DNA breaks earlier than other known proteins, and thus is useful in methods for readily and easily visualizing the presence of DNA breaks as evidence of DNA damage in cells. Additionally, as demonstrated below, concentrated foci of 53Bp1 are detectable in several genetically unstable tumor cell lines, thereby enabling this protein to be a useful target for development and identification of novel cancer therapeutic agents, and possibly the delivery of other therapeutic agents.

[0031] By the term “biological activity of 53Bp1” as used herein, is meant the ability to localize quickly to DNA DSBs, as well as to localize in discrete foci in certain tumor cells. The other characteristics of 53Bp1, identified by the inventors, are discussed throughout this specification and included in this definition. See, Examples 1 through 7, that demonstrate other characteristics of the 53Bp1 protein.

[0032] The known encoding nucleic acid sequence (SEQ ID NO: 1) and protein sequence (SEQ ID NO: 2) of human 53Bp1 are provided in GenBank, Accession No. AF078776, submitted Jul. 16, 1998. As used herein, the term “53Bp1 nucleic acid sequence” refers not only to the isolated nucleic acid segment or fragment reported in SEQ ID NO: 1, which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, such as the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0033] When used in the compositions and methods of this invention, the term 53Bp1 can include fragments of the protein (or of nucleotide sequences encoding the protein). Such fragments may include fragments that are minimally necessary for targeting the 53Bp1 to the site of DNA damage. As taught in Example 9, one such fragment may be that spanning amino acid residues 1220 to 1711 of SEQ ID NO: 2. Another fragment is a deletion mutant which is missing an unnecessary portion of the 53Bp1 sequence. For example, one such deletion mutant fragment spans amino acid residues 1 to 1711 of SEQ ID NO: 2, or a fusion of AA residues 1-1053 with 1220-1972, or AA residues 1-34 with AA residues 1047-1972, or AA residues 1-34 with AA residues 1047-1711, or AA residues 1-34 with AA residues 1220-1711 of SEQ ID NO: 2. Still other fragments of 53Bp1 which contain only necessary targeting sequences, e.g., more than the minimal sequence AA 1220 to 1711 of SEQ ID NO: 2 can be designed and used by one of skill in the art given the teachings herein.

[0034] When used in the compositions and methods of this invention, the isolated nucleic acid of 53Bp1, or fragments of that sequence encoding minimal targeting fragments of 53Bp1, should not be construed as being limited solely to the known nucleotide sequences of SEQ ID NO: 1, but rather should be construed to include any and all nucleotide sequences which share homology (i.e., have sequence identity) with that nucleotide sequence. Preferably, the invention includes an isolated nucleic acid having a nucleotide sequence which is at least 70% identical to the nucleotide sequence presented in SEQ ID NO: 1. More preferably, an isolated nucleic acid of this invention has a nucleotide sequence which is at least 75% identical, even more preferably, 80% identical, yet more preferably, 85% identical, and even more preferably, 90% identical to the nucleotide sequence presented in SEQ ID NO: 1. Even more preferably, an isolated nucleic acid of this invention has a nucleotide sequence which is at least 95% identical, and most preferably, 99% identical, to the nucleotide sequence presented in SEQ ID NO: 1. Any such isolated nucleic acid would of course encode a polypeptide having the biological activity of 53Bp1, as disclosed herein.

[0035] “Homologous” as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3′ ATTGCC 5′ and 3′ TATGGC 5′ share 50% homology. As used herein, “homology” is used synonymously with “identity”.

[0036] Percent identity, percent similarity or percent homology of one polynucleotide or polypeptide with respect to another identified polynucleotide or polypeptide is calculated using algorithms, such as the Smith-Waterman algorithm [J. F. Collins et al, 1988, Comput. Appl. Biosci., 4:67-72; J. F. Collins et al, Molecular Sequence Comparison and Alignment, (M. J. Bishop et al, eds.) In Practical Approach Series: Nucleic Acid and Protein Sequence Analysis XVIII, IRL Press: Oxford, England, UK (1987) pp.417], and the BLAST and FASTA programs [E. G. Shpaer et al, 1996, Genomics, 38:179-191]. A preferred algorithm is the computer program BLAST, especially blastp or tblastn (Altschul et al., 1997). These references are incorporated herein by reference. Sequence homology for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. Unless otherwise specified, the parameters of each algorithm discussed above are the default parameters identified by the authors of such algorithms.

[0037] Among such homologous nucleotide sequences of this invention are allelic variants of the 53Bp1 sequence of SEQ ID NO: 1 within a species (i.e., sequences containing some individual nucleotide differences from a more commonly occurring sequence within a species, but which nevertheless encode the same polypeptide or a protein with the same function). Additionally nucleic acid sequences capable of hybridizing under stringent conditions [see, J. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory (1989)] to the sequences of SEQ ID NO: 1, their anti-sense strands, or biologically active fragments thereof are homologous sequences according to this invention. An example of a highly stringent hybridization condition is hybridization at 2×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for an hour. Alternatively, an exemplary highly stringent hybridization condition is in 50% formamide, 4×SSC at 42° C. Moderately high stringency conditions also prove useful, e.g., hybridization in 4×SSC at 55° C., followed by washing in 0.1×SSC at 37° C. for an hour. An alternative exemplary moderately high stringency hybridization condition is in 50% formamide, 4×SSC at 30° C.

[0038] Depending upon its use in the methods and compositions of this invention, the known 53Bp1 nucleic acid sequence [SEQ ID NO: 1] is modified. Utilizing the known sequence, useful modifications are within the skill of the art, e.g., synthetic or recombinant polynucleotide sequences, or modified polynucleotide sequences, encoding the full-length 53Bp1 protein or useful fragments thereof. Such modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences which are silent or which change the amino acids, e.g. to improve expression or secretion. Also included are allelic variations, caused by the natural degeneracy of the genetic code. Additional homologous sequences can include mutants including 5′ or 3′ terminal or internal deletions, which truncated or deletion mutant sequence are expressed for the purpose of affecting the activity of the full-length or wild-type 53Bp1 polypeptide or fragments.

[0039] Similarly, the term 53Bp1 protein or polypeptide, or fragments encompassing the minimal targeting fragment, as used herein should not be construed as being limited solely to the known amino acid sequence of SEQ ID NO: 2, but rather should be construed to include any and all amino acid sequences which share homology (i.e., have sequence identity) with those amino acid sequences. Preferably, the methods and compositions of this invention make use of a polypeptide having an amino acid sequence which is 70% identical, more preferably, 75% identical, even more preferably, 80% identical, yet more preferably, 85% identical, even more preferably, 90% identical, more preferably, 95% identical and most preferably, 99% or 100% identical to the known amino acid sequence presented in SEQ ID NO: 2. Reference to 53Bp1 herein includes the definitions of “homologous”, “homology” and “percent identity” as discussed above, including the list of computer algorithms available to calculate these homologies. Any such preparation of a homologous polypeptide has the biological activity of the 53Bp1 as disclosed herein.

[0040] Also included in the invention are modified versions of the 53Bp1 polypeptide. Typically, such polypeptides differ from the known 53Bp1 polypeptide of SEQ ID NO: 2 by only one to four codon changes. Examples include polypeptides with minor amino acid variations from the known amino acid sequence of 53Bp1 (SEQ ID NO: 2), in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. Further encompassed by this invention are compositions and methods employing fragments of 53Bp1, including fragments containing the minimal targeting sequences identified above. Useful fragments are designed or obtained in any desired length, including as small as about 5-8 amino acids in length, or larger fragments, such as about 490 amino acids or more. Such fragments are desirably characterized by localizing to the sites of DNA DSBs or having a biological activity similar to the intact 53Bp1.

[0041] B. Methods of Preparing Sequences of this Invention

[0042] Methods for obtaining the nucleic acids and polypeptides of the invention should be apparent to those skilled in the art given the present disclosure and the instructions known to one of skill in the art. For example, the nucleotide and polypeptide sequences useful in the compositions and methods of the invention are prepared conventionally by resort to known chemical synthesis techniques, e.g., solid-phase chemical synthesis, such as described by Merrifield, 1963 J. Amer. Chem. Soc., 85:2149-2154, and J. Stuart and J. Young, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, Ill. (1984), or detailed in the examples below.

[0043] Alternatively, the nucleotide and polypeptide sequences useful in the methods and compositions of this invention are prepared by known recombinant DNA techniques and genetic engineering techniques, such as polymerase chain reaction, by cloning and expressing within a host microorganism or cell a DNA fragment carrying a nucleic acid sequence encoding the above-described polypeptides, etc. [See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual., 2d Edit., Cold Spring Harbor Laboratory, New York (1989); Ausubel et al. (1997), Current Protocols in Molecular Biology, John Wiley & Sons, New York]. The 53Bp1 are obtained from gene banks derived from whole genomic DNA. These sequences, fragments thereof, modifications thereto and the full-length sequences are constructed recombinantly using conventional molecular biology techniques, site-directed mutagenesis, genetic engineering or PCR, and the like by utilizing the information provided herein. For example, methods for producing the above-identified modifications of the sequences, include mutagenesis of certain nucleotides and/or insertion or deletion of nucleotides, or codons, thereby effecting the polypeptide sequence by insertion or deletion of, e.g., non-natural amino acids, are known and selected by one of skill in the art.

[0044] 1. Expression In Vitro

[0045] To produce recombinant 53Bp1 or other fragments of this invention in vitro, the DNA sequences of the invention are inserted into a suitable expression system. Desirably, a recombinant molecule or vector is constructed in which the polynucleotide sequence encoding the selected protein is operably linked to a heterologous expression control sequence permitting expression of the protein. Numerous types of appropriate expression vectors are known in the art for protein expression, by standard molecular biology techniques. Such vectors are selected from among conventional vector types including insects, e.g., baculovirus expression, or yeast, fungal, bacterial or viral expression systems. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose. Methods for obtaining such expression vectors are well-known. See, Sambrook et al, cited above; Miller et al, 1986 Genetic Engineering, 8:277-298 and references cited therein.

[0046] Suitable host cells or cell lines for transfection by this method include bacterial cells. For example, the various strains of E. coli (e.g., HB101, MC1061, and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, Streptomyces, and other bacilli and the like are also employed in this method. Mammalian cells, such as human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NIH mice are used. Another suitable mammalian cell line is the CV-1 cell line. Still other suitable mammalian host cells, as well as methods for transfection, culture, amplification, screening, production, and purification are known in the art. [See, e.g., Gething and Sambrook, 1981 Nature, 293:620-625, or alternatively, Kaufman et al, 1985 Mol. Cell. Biol., 5(7):1750-1759 or Howley et al, U.S. Pat. No. 4,419,446]. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Other fungal cells are also be employed as expression systems. Alternatively, insect cells such as Spodoptera frugipedera (Sf9) cells may be used.

[0047] Thus, the present invention provides a method for producing a recombinant 53Bp1 protein, which involves transfecting, e.g., by conventional means such as electroporation, a host cell with at least one expression vector containing a polynucleotide of the invention under the control of a transcriptional regulatory sequence. The transfected or transformed host cell is then cultured under conditions that allow expression of the protein. The expressed protein is recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art. For example, the proteins are isolated in soluble form following cell lysis, or extracted using known techniques, e.g., in guanidine chloride. If desired, the proteins or fragments of the invention are produced as a fusion protein to enhance expression of the protein in a selected host cell, to improve purification, or for use in monitoring the presence of the desired protein in tissues, cells or cell extracts. Suitable fusion partners for the proteins of the invention are well known to those of skill in the art and include, among others, β-galactosidase, glutathione-S-transferase, and poly-histidine.

[0048] 2. Expression In Vivo

[0049] Alternatively, where it is desired that the 53Bp1 protein or ligand useful in the methods and compositions of the invention or proteinaceous inhibitors thereof (whether full-length or a desirable fragment) be expressed in vivo, e.g., to induce antibodies, or as a therapeutic, an appropriate vector for delivery is readily selected by one of skill in the art. Exemplary vectors for in vivo gene delivery are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus [International patent application No. PCT/US91/03440], adenovirus vectors [M. Kay et al, 1994 Proc. Natl. Acad. Sci. USA, 91:2353; S. Ishibashi et al, 1993 J. Clin. Invest., 92:883], or other viral vectors, e.g., various poxviruses, vaccinia, etc. Methods for insertion of a desired gene, e.g., P7-1, and obtaining in vivo expression of the encoded protein, are well known to those of skill in the art.

[0050] The preparation or synthesis of the nucleotide and polypeptide sequences, including the ligands disclosed herein, whether in vitro or in vivo (including ex vivo) is well within the ability of the person having ordinary skill in the art using available material. The synthetic methods are not a limitation of this invention.

[0051] C. Ligands and Inhibitors of 53Bp1

[0052] Based on the information on the biological activities of 53Bp1 identified by the inventors, the present invention provides in one aspect, compositions that can inhibit the expression of the protein and hence prevent its biological function, as well as compositions that bind to the protein and antagonize, inhibit or block the biological functions of 53Bp1.

[0053] Such compositions have utility as diagnostic reagents or as therapeutic reagents in the methods described below. By the use of the term “53Bp1 ligand or 53Bp1 protein ligand” as used herein is meant a compound, e.g., an antibody, which is capable of detecting the formation of concentrated 53Bp1 nuclear foci in cells exposed to agents which cause DNA DSBs by binding to some characteristic portion or epitope of 53Bp1. Such ligands are additionally characterized as antagonizing or inhibiting the biological function of 53Bp1. By the term “53Bp1 inhibitor” is meant a composition which inhibits or prevents the expression of 53Bp1, such as an antisense sequence which binds to the 53Bp1 messenger RNA, and thereby inhibits or prevents the biological function of 53Bp1. Inhibition of 53Bp1 activity by either a 53Bp1 ligand or a 53Bp1 inhibitor is assessed by following the procedures presented in the examples herein, which permit the formation of foci or lack of such formation to be detected.

[0054] 1. Nucleotide Sequence 53Bp1 Inhibitors

[0055] One such 53Bp1 inhibitor is a nucleotide sequence that binds to the 53Bp1 nucleic acid sequence or a fragment thereof, preferably the 53Bp1 mRNA. For example, such a 53Bp1 inhibitor includes an oligonucleotide molecule which is preferably in an antisense orientation with respect to the nucleic acid sequence of 53Bp1. As used herein, the term “antisense oligonucleotide” means a nucleic acid polymer, at least a portion of which is complementary to a 53Bp1 mRNA or other nucleic acid, particularly the BRCT domains. “Antisense” refers particularly to the nucleic acid sequence of the noncoding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence is complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

[0056] The antisense oligonucleotides of the invention preferably comprise between about fourteen and about fifty nucleotides. More preferably, the antisense oligonucleotides comprise between about twelve and about thirty nucleotides. Most preferably, the antisense oligonucleotides comprise between about sixteen and about twenty-one nucleotides. The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides. Methods for synthesizing oligonucleotides, phosphorothioate oligonucleotides, and otherwise modified oligonucleotides are well known in the art [U.S. Pat. No. 5,034,506; Nielsen et al., 1991, Science 254:1497].

[0057] By binding to the mRNA of 53Bp1, the antisense sequence inhibits expression of the 53Bp1 protein, and thereby prevents or inhibits the 53Bp1 biological DNA repair function.

[0058] 2. Polypeptide/Protein Ligands

[0059] In another embodiment, another ligand composition of the invention binds to the 53Bp1 polypeptide. Such ligands, when contacted with a cell exposed to a DNA damaging agent which results in DNA DSBs, e.g., radiation, cancer, etc. can locate 53Bp1 foci in that cell. Such a ligand is desirably an antibody which binds to 53Bp1, e.g., the BRCT domains or other unique domains of 53Bp1. Preferably, such a ligand also inhibits 53Bp1 biological function. The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to an epitope on 53Bp1, e.g., the BRCT domain. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention exist in a variety of forms including, for example, high affinity polyclonal antibodies, monoclonal antibodies, synthetic antibodies, chimeric antibodies, recombinant antibodies and humanized antibodies. Such antibodies originate from immunoglobulin classes IgG, IgM, IgA, IgD and IgE. One such desirable ligand is the anti-53Bp1 monoclonal antibody described in detail in Example 1. Other such antibodies include a Fab, Fab′ or F(ab′)2, or Fc antibody fragment thereof which binds 53Bp1. Still another useful ligand is a single chain Fv antibody fragment which binds 53Bp1.

[0060] Another useful ligand is a recombinant construct comprising a complementarity determining region of an antibody, a synthetic antibody or a chimeric antibody construct which shares sufficient CDRs to retain functionally equivalent binding characteristics of an antibody that binds 53Bp1. By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0061] The antibodies of this invention are generated by conventional means utilizing the isolated, recombinant or modified 53Bp1 or fragments thereof as antigens of this invention. For example, polyclonal antibodies are generated by conventionally stimulating the immune system of a selected animal or human with a 53Bp1 antigen, allowing the immune system to produce natural antibodies thereto, and collecting these antibodies from the animal or human's blood or other biological fluid. Preferably a recombinant version of 53Bp1 is used as an immunogen. Monoclonal antibodies MAbs) directed against 53Bp1 are also generated conventionally. Hybridoma cell lines expressing desirable MAbs are generated by well-known conventional techniques, e.g. Kohler and Milstein and the many known modifications thereof. Similarly desirable high titer antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens [see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al., 1989 Proc. Nat'l. Acad. Sci. USA, 86:10029-10033; PCT Patent Application No. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988); Huse et al, 1988a Science, 246:1275-1281].

[0062] Given the disclosure contained herein, one of skill in the art generates ligands or antibodies directed against 53Bp1 by resort to known techniques by manipulating the complementarity determining regions of animals or human antibodies to the antigen of this invention. See, e.g., E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer-Verlag (June, 1994); Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Bird et al., 1988, Science 242:423-426.

[0063] Alternatively, 53Bp1 antigens are assembled as multi-antigenic complexes [see, e.g., European Patent Application 0339695, published Nov. 2, 1989] and employed to elicit high titer antibodies capable of binding the 53Bp1. Further provided by the present invention are anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3). Ab2 are specific for the target to which anti-53Bp1 antibodies of the invention bind and Ab3 are similar to 53Bp1 antibodies (Ab1) in their binding specificities and biological activities [see, e.g., M. Wettendorff et al., “Modulation of anti-tumor immunity by anti-idiotypic antibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux, 1990 J. Am. Soc. Microbiol., Washington D.C.: pp. 203-229). These anti-idiotype and anti-anti-idiotype antibodies are produced using techniques well known to those of skill in the art. Such anti-idiotype antibodies (Ab2) can bear the internal image of 53Bp1 and are thus useful for the same purposes as 53Bp1.

[0064] In general, polyclonal antisera, monoclonal antibodies and other antibodies which bind to 53Bp1 as the antigen (Ab1) are useful to identify epitopes of 53Bp1 to separate 53Bp1 and its analogs from contaminants in living tissue (e.g., in chromatographic columns and the like), and in general as research tools and as starting material essential for the development of other types of antibodies described above. Anti-idiotype antibodies (Ab2) are useful for binding the same target and thus are used in place of 53Bp1 to induce useful ligands to 53Bp1. The Ab3 antibodies are useful for the same reason the Ab1 are useful. Other uses as research tools and as components for separation of 53Bp1 from other contaminants, for example, are also contemplated for the above-described antibodies.

[0065] Other ligands include small chemical compounds that bind to 53Bp1 and prevents its ability to form nuclear foci. Still other chemical compounds bind to 53Bp1 and prevent its ability to participate in DNA repair. Such 53Bp1 ligands are identified and developed by the drug screening methods discussed in detail below.

[0066] 3. Ligands/Inhibitors as Diagnostic Reagents and Kits

[0067] For use in diagnostic assays and kits for the detection of DNA DSBs caused by exposure to DNA damaging agents, the above-described inhibitors or ligands of 53Bp1 are preferably associated with a detectable label which is capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Where more than one 53Bp1 inhibitor or ligand is employed in a diagnostic method, the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. by fluorescence, for ready use in immunohistochemical analyses or immunofluorescent microscopy. Preferably, each inhibitor or ligand is associated with, or conjugated to a fluorescent detectable fluorochromes, e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). All of these fluorescent dyes are commercially available, and their uses known to the art. Other useful labels include a colloidal gold label.

[0068] Still other useful labels include radioactive compounds or elements. Additionally, labels include a variety of enzyme systems that operate to reveal a calorimetric signal in an assay, e.g., glucose oxidase (which uses glucose as a substrate) releases peroxide as a product which in the presence of peroxidase and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color. Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase in conjunction with glucose-6-phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength.

[0069] Other label systems that are utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles [Bangs Laboratories, Indiana] in which a dye is embedded are used in place of enzymes to form conjugates with the inhibitor sequences or ligands and provide a visual signal indicative of the presence of the resulting complex in applicable assays.

[0070] Detectable labels for attachment to 53Bp1 inhibitors or ligands and antibodies useful in diagnostic assays of this invention are easily selected from among numerous compositions known and readily available to one skilled in the art of diagnostic assays. The diagnostic reagents, e.g., the inhibitors and ligands of this invention, are not limited by the particular detectable label or label system employed.

[0071] Methods for coupling or associating the label with the inhibitor or ligand are similarly conventional and known to those of skill in the art. Known methods of label attachment are described [see, for example, Handbook of Fluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland, Molecular Probes, Inc., Eugene, Oreg., 1996; Pierce Catalog and Handbook, Life Science and Analytical Research Products, Pierce Chemical Company, Rockford, Ill., 1994/1995]. Thus, selection of the label and coupling methods do not limit this invention.

[0072] For convenience, the conventional reagents for immunohistochemical analysis or immunofluorescent microscopy, or other diagnostic assays according to this invention are provided in the form of kits. Such kits are useful for determining and enumerating the absence or presence of 53Bp1 foci in a cell or tissue, particularly a tumor cell. Thus, such a kit will be useful in conducting the diagnostic assays discussed below, e.g., in determining if a cell is cancerous, in determining the status of cells or tissues exposed to DNA damage, etc. Such a diagnostic kit contains a nucleotide inhibitor (e.g., a 53Bp1 antisense sequence), or 53Bp1 ligand (e.g., an antibody capable of binding 53Bp1) of this invention. Alternatively, such kits contain a simple mixture of such inhibitors or means for preparing a simple mixture. The kits also include instructions for performing the assay, microscopic slides for fixing the tissue or cells, fixatives, suitable stains, or microtiter plates to which the inhibitors or nucleic acid sequences of the invention have been pre-adsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound compositions and other signal-generating reagents, such as fluorescent compounds and dyes, enzyme substrates, cofactors and chromogens. Other components include indicator charts for fluorescent or calorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, and a sample preparator cup. Such kits provide a convenient, efficient way for a clinical laboratory to diagnose the presence or absence of DNA damage in a cell or tissue according to this invention.

[0073] 4. Inhibitors/Ligands as Therapeutic Compositions of this Invention

[0074] Alternatively, an above-described inhibitor or ligand of 53Bp1 of this invention which antagonizes or inhibits the biological activity of 53Bp1, or which binds to the 53Bp1, is employed therapeutically, and as such, is encompassed in a pharmaceutical composition for treating cancers that are characterized by 53Bp1 nuclear foci. Such a composition includes a 53Bp1 ligand or inhibitor (nucleotide or polypeptide or protein) and a pharmaceutically-acceptable carrier. As used herein, the term “pharmaceutically-acceptable carrier” means a chemical composition with which an appropriate 53Bp1 inhibitor or ligand is combined and which, following the combination, is used to administer the appropriate 53Bp1 inhibitor or ligand to a mammal. Typical carriers include saline, buffered saline, and other inert compositions known and used in drug delivery. In addition to the appropriate 53Bp1 inhibitor or ligand, such pharmaceutical compositions optionally also contain other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems are useful to administer an appropriate 53Bp1 inhibitor or ligand according to the methods of the invention.

[0075] Also, as noted herein, pharmaceutical compositions of this invention include a combination of compounds comprising a 53Bp1 ligand or inhibitor associated with another chemotherapeutic, which functions to kill or retard the growth of the cell containing 53Bp1 foci.

[0076] Still other compositions that inhibit 53Bp1 functions, such as compositions, synthetic compounds or other compounds identified as 53Bp1 ligands or inhibitors by the screening methods described below are optionally employed in pharmaceutical compositions for treating cancer.

[0077] Pharmaceutical therapeutic or vaccinal compositions that are useful in the methods of the invention are administered systemically in oral solid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations which are administered intravenously, intraperitoneally, subcutaneously or by other routes known for pharmaceutical administration. Selection of the formulations and routes are within the skill of the art, and are not a limitation of this invention.

[0078] 5. Research Uses for 53Bp1 Ligands

[0079] The above-described ligands are useful as research tools for categorizing tumor cells or other cancer cells which develop 53Bp1 foci in the absence of DNA damaging agents from normal cells or from cancer cells which develop such foci only after such exposure. Such ligands are useful in drug modeling in the methods disclosed below.

[0080] D. Diagnostic Methods of the Invention

[0081] Based on the novel biological activities of 53Bp1 as determined by the inventor, another embodiment of this invention is a method for simply and rapidly detecting DNA damage in a biological sample utilizing the above-mentioned ligands of 53Bp1. By “biological sample” is meant any mammalian cell or tissue, or cell or tissue-containing composition or isolate. For example, one biological sample may be a cell scraping, exudate or tissue specimen for biopsy, e.g., a buccal sample, sputum, cervical scraping. Another type of biological sample may be a preparation containing white blood cells, e.g., peripheral blood, sputum, saliva, urine, etc. for use in detecting the presence or absence of DNA damage in a patient that has been exposed to a DNA DSB inducing agent, such as radiation, chemicals, etc. Thus, the diagnostic method of this invention comprises contacting the biological sample, preferably immobilized or fixed on a surface, such as a microscope slide, with a ligand that binds to human 53Bp1. Such ligands are discussed in detail above, and are preferably associated with a label which provides a detectable signal, also as discussed above. The sample is then examined for the presence of signal concentrated in nuclear foci of 53Bp1 in the cells of the sample. The examining step is any suitable assay step, including, without limitation, fluorescent immunomicroscopy or immunohistochemical analysis.

[0082] The presence of concentrated foci is indicative of DNA damage, while the presence of diffuse signal is indicative of a lack of DNA damage in the sample. Thus, this method is used to rapidly and easily identify cancer cells in conventional cancer screening and is used to monitor the status of anti-cancer therapies. Additionally, this method is also employed to rapidly and readily assess the possibility of DNA damage in patients exposed to gamma irradiation or other DNA damage agents, particularly those known to cause DNA DSBs.

[0083] E. Drug Screening Methods of the Invention

[0084] Methods of screening test compounds are described which can identify a composition that either binds to 53Bp1, and is thus useful as a targeting agent for association with a chemotherapeutic agent, or a composition that binds to and inhibits or antagonizes the biological activity of 53Bp1 directly, and is thus useful as a direct chemotherapeutic. One such screening method can readily utilize the methods outlined in the examples below. For example, one method comprises employing a 53Bp1 ligand associated with a detectable label to detect the expression of 53Bp1 in a cell contacted with a test compound or to detect the presence or number of 53Bp1 induced nuclear foci in cells contacted with a test compound. Such a method involves contacting a selected cell with a test compound, and then exposing the selected cell and test compound (i.e., the “test cell”) as well as an identical cell without test compound (i.e., the “control cell”) to a DNA damaging agent, such as gamma irradiation. The test cell and control cell are then exposed to a 53Bp1 labeled ligand, such as a fluorescently-labeled anti-53Bp1. Because 53Bp1 nuclear foci form quickly after exposure of the cell to the damaging agent, the test cell and the control cell are then examined for the presence and number of 53Bp1 foci by a technique such as immunofluorescent microscopy. The results of such examination are then compared. The absence of foci in the test cell (or a significant reduction in the number of such foci) when compared to the control cell (which should have a significant number of foci) is an indication that the test compound inhibited the biological activity of 53Bp1 in this assay. The presence and/or number of foci is indicated by the level or intensity of the signal generated by the label on the ligand. The signal (or its level of expression or intensity) indicates the presence and number of 53Bp1 nuclear foci. When the signals generated by the label in the tests cell are compared with the signals (if any) generated by the labels in the control cell, a lesser detectable signal in the test cell indicates that said test compound has inhibited the presence and/or number of 53Bp1 foci in the cell (a) and is, in fact, a 53Bp1 inhibitor. Similar assays using different ligands, different detection techniques, etc. are readily designed by one of skill in the art with resort to the information provided in the art generally and in the examples below.

[0085] Inhibitors of 53Bp1 activity are screened by resort to assays and techniques useful in identifying drugs capable of binding to the 53Bp1 polypeptide and thereby inhibiting its biological activity in a cancer cell that expresses 53Bp1 in the absence of DNA damaging agents. Such assays include the use of phage display system for expressing the 53Bp1 polypeptide, and using a culture of transfected E. coli or other microorganism to produce the proteins for binding studies of potential binding compounds. See, for example, the techniques described in G. Cesarini, 1992 FEBS Letters, 307(1):66-70; H. Gram et al., 1993 J. Immunol. Meth., 161:169-176; C. Summer et al., 1992 Proc. Natl. Acad. Sci., USA, 89:3756-3760, incorporated by reference herein.

[0086] Other conventional drug screening techniques are employed using the proteins, antibodies or polynucleotide sequences of this invention. As one example, a method for identifying compounds which specifically bind to a 53Bp1 polypeptide of this invention can include simply the steps of contacting a selected cell expressing 53Bp1 with a test compound to permit binding of the test compound to 53Bp1 and determining the amount of test compound, if any, which is bound to the 53Bp1. Such a method involves the incubation of the test compound and the 53Bp1 polypeptide immobilized on a solid support. Typically, the surface containing the immobilized ligand is permitted to come into contact with a solution containing the protein and binding is measured using an appropriate detection system. Suitable detection systems include those described above for diagnostic use.

[0087] Thus, through use of such methods, the present invention is anticipated to provide compounds capable of interacting with 53Bp1 or portions thereof, and either enhancing or decreasing 53Bp1's biological activity, as desired. Such compounds are believed to be encompassed by this invention.

[0088] Still other methods of drug screening for novel compounds that inhibit 53Bp1 expression at the nucleic acid or protein level involve computational evaluation and design. According to these methods, the three dimensional structure of the 53Bp1 gene and/or the polypeptide is determined and chemical entities or fragments are screened and selected for their ability to associate with the three dimensional structures. Suitable software for such analysis include docking software such as Quanta and Sybyl, molecular dynamics and mechanics programs, such as CHARMM and AMBER, the GRID program available from Oxford University, Oxford, UK. [P. J. Goodford, “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules”, 1985 J. Med. Chem., 28:849-857]; the MCSS program available from Molecular Simulations, Burlington, Mass. [A. Miranker and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method”, 1991 Proteins: Structure, Function and Genetics, 11:29-34]; the AUTODOCK program available from Scripps Research Institute, La Jolla, Calif. [D. S. Goodsell and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing”, 1990 Proteins: Structure, Function, and Genetics, 8:195-202]; and the DOCK program available from University of California, San Francisco, Calif. [I. D. Kuntz et al, “A Geometric Approach to Macromolecule-Ligand Interactions”, 1982 J. Mol. Biol., 161:269-288]. Additional commercially available computer databases for small molecular compounds include Cambridge Structural Database, Fine Chemical Database, and CONCORD database [for a review see Rusinko, A., Chem. Des. Auto. News, 8:44-47 (1993)].

[0089] Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or 53Bp1 inhibitor. Assembly may proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the 3D structure of 53Bp1. This would be followed by manual model building using software such as Quanta or Sybyl software, CAVEAT program [P. A. Bartlett et al, 1989 “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules”, in Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78, pp. 182-196], which is available from the University of California, Berkeley, Calif.; 3D Database systems such as MACCS-3D database (MDL Information Systems, San Leandro, Calif.) [see, e.g., Y. C. Martin, “3D Database Searching in Drug Design”, 1992 J. Med Chem., 35:2145-2154]; and the HOOK program, available from Molecular Simulations, Burlington, Mass.

[0090] Other molecular modeling techniques are employed in accordance with this invention. See, e.g., N. C. Cohen et al, “Molecular Modeling Software and Methods for Medicinal Chemistry”, 1990 J. Med. Chem., 33:883-894. See also, M. A. Navia and M. A Murcko, “The Use of Structural Information in Drug Design”, 1992 Current Opinions in Structural Biology, 2:202-21. For example, where the structures of test compounds are known, a model of the test compound is superimposed over the model of the structure of the invention. Numerous methods and techniques are known in the art for performing this step, any of which may be used. See, e.g., P. S. Farmer, Drug Design, Ariens, E. J., ed., Vol. 10, pp 119-143 (Academic Press, New York, 1980); U.S. Pat. No. 5,331,573; U.S. Pat. No. 5,500,807; C. Verlinde, 1994 Structure, 2:577-587; and I. D. Kuntz, 1992 Science, 257:1078-1082. The model building techniques and computer evaluation systems described herein are not a limitation on the present invention.

[0091] Thus, using these computer evaluation systems, a large number of compounds are quickly and easily examined and expensive and lengthy biochemical testing avoided. Moreover, the need for actual synthesis of many compounds is effectively eliminated. Once identified by the modeling techniques, the 53Bp1 inhibitors identified by these methods is tested for bioactivity using the assays described herein.

[0092] F. Pharmaceutical Methods of the Invention

[0093] As indicated by the examples below, certain cancer cells develop DNA DSBs and consequently 53Bp1 nuclear foci in the absence of known DNA damaging agents. Still other cells may become cancerous and develop such 53Bp1 foci only after exposure to DNA damaging agents. As another aspect, this invention provides a method for retarding the growth of or killing a cancer cell by administering to the site of a cancer cell a 53Bp1 inhibitor or ligand. Such an inhibitor antagonizes or inhibits the biological activity of the 53Bp1 directly and results in tumor cell death. In such a method, the administration of the inhibitor to the cell occurs ex vivo, e.g., for cells which are desired to be purged of cancer cells and returned to the patient, e.g., blood. Alternatively, the patient is treated in vivo by administering the ligand inhibitor in a suitable pharmaceutical preparation directly to a mammal having a cancer.

[0094] Still another pharmaceutical method involves using the 53Bp1 inhibitor to indirectly treat the tumor cell by binding to the 53Bp1 mRNA and inhibiting or preventing the expression of the protein. If the protein is inhibited, it cannot migrate to the site of DNA damage. The absence of the repair function of 53Bp1 makes the tumor cell more sensitive to damage by other, conventional chemotherapeutics, such as RicinA, toxins, bispecific antibodies associated with host protective cells, anticancer drugs, such as doxorubicin or 5-FU, optionally linked to another protein or ligand, among others.

[0095] In yet a further pharmaceutical regimen, the 53Bp1 ligand, such as a low affinity antibody or other ligand that preferentially binds to 53Bp1 foci, targets the tumor cell for delivery of another therapeutic agent, such as the agents identified above or other chemotherapeutic agents. In this method, the ligand is associated to a second compound that retards the growth of, or kills, the tumor cell, once it is delivered to the site of 53Bp1 foci by the binding of the ligand to the 53Bp1. The second compound includes, without limitation, a radionucleotide, a toxin, a bi-specific antibody and an anticancer drug optionally linked to a protein or peptide. The ligand with its associated chemotherapeutic compound, in a suitable pharmaceutical carrier, is delivered ex vivo or in vivo to the mammalian tissue. For example, the ligand is administered directly to a patient bearing tumor cells containing 53Bp1 foci or other cancer cells containing 53Bp1 foci.

[0096] These pharmaceutical compositions described above are administered via any suitable therapeutic route, and selection of such route is not a limitation of this invention. Similarly the appropriate dosage of such pharmaceutical compositions are determined by a physician, based on typical characteristics such as the physical condition of the patient, the disease being treated, the identity of the associated therapeutic or the subsequent or simultaneous uses of other therapeutic compositions, etc. In one embodiment, the pharmaceutical compositions useful for practicing the therapeutic methods of the invention are administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day of the ligand. The dosage of the ligand is determined, adjusted and prepared for booster administrations, if any, by one of skill in the art. This invention is not limited by the dosage selection.

[0097] The following examples illustrate several embodiments of this invention. The following examples illustrate the function of 53Bp1 as a DNA damage-responsive protein in cycling cells, and demonstrate that 53Bp1 participates in the maintenance of genome integrity. These examples are illustrative only, and do not limit the scope of the present invention.

EXAMPLE 1 Experimental Procedures

[0098] A. Antibodies.

[0099] High affinity monoclonal antibodies, called anti-53BP1, were generated to be directed against the carboxy terminal BRCT domain of 53BP1. The monoclonal antibodies were generated by the conventional techniques described above, such as immunizing an animal using as the antigen a purified recombinant fragment of 53Bp1 encompassing the C-terminal 312 amino acids of 53Bp1 (amino acids 1662 to 1973 of SEQ ID NO: 2) which includes the BRCT domain (i.e., amino acids 1713 to 1973 of SEQ ID NO: 2). Other fragments of 53Bp1 are also used to make anti-53Bp1 antibodies by the published methods cited above. These antibodies were used in the examples below to probe the intracellular localization of 53Bp1.

[0100] Other antibodies used in the examples below are anti-Mre11 and p95/NBS, polyclonal antibodies (Calbiochem, San Diego, Calif.); anti Rad50, a polyclonal antibody (obtained from John Petrini at the University of Wisconsin); and Y11, a polyclonal antibody that recognizes the N-terminal hemaglutinin (HA) tags (Santa Cruz Biotech, Santa Cruz, Calif.).

[0101] B. Cell Lines.

[0102] Stably transfected U2OS cell lines were created by transfecting the plasmid pSV2 [Chehab, N. H. et al, 1999 Proc. Natl. Acad. Sci., USA, 96:13777-13782] containing various N-terminal hemaglutinin (HA) tagged versions of 53Bp1. HA53Bp1 is the full-length protein bearing the HA tag on its N terminus. HA53Bp1ΔBRCT is similar to HA53Bp1 with amino acids 1713 to 1973 (the BRCT domains) of SEQ ID NO: 2 deleted therefrom. HA53Bp1C312 contains only the C-terminal 312 amino acids of 53Bp1, which include the nuclear localization signal (i.e., amino acids 1662 to 1701 of SEQ ID NO: 2 and the BRCT domain (amino acids 1702-1973 of SEQ ID NO: 2). The U2OS cells on cover slips were transiently transfected with the selected plasmid using the calcium phosphate method. Positively transfected colonies were pooled after selecting with neomycin. Expression was allowed to occur for 30 hours and then cells were treated, fixed, stained, and visualized as described below.

[0103] C. Analysis of Ionizing Radiation-Induced Foci by Immunofluorescent Microscopy.

[0104] For use in the specifically defined experiments below, U20S cells grown on cover slips were mock treated for use as controls or the cells were exposed to DNA damaging agents, such as 50 J/m² UV light, 0.2 mM etoposide, 1 mM hydroxyurea, or between 0.5 and 12 Gy gamma irradiation via a ¹³⁷Cs-irradiator. In some experiments, the cells were exposed to 20 μM of the fungal metabolite wortmannin, a known inhibitor of DNA-dependent protein kinase (DNA-PK) and double strand break rejoining, for 1 hour prior to irradiation. The wortmannin remained on cells for all time points. Five minutes to 24 hours later cells were fixed in 1% paraformaldehyde for 15 minutes, followed by extraction on ice for 20 minutes in 0.2% Triton X-100/phosphate buffered saline (PBS). Cover slips were incubated with the selected antibody for 1 hour at room temperature, washed with PBS and exposed to anti-mouse immunoglobulin (IgG) conjugated to the fluorescent dye, Texas Red (Vector Labs) or anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) for 30 minutes at room temperature. After washing, the cells were counterstained with DAPI, washed, and mounted on glass slides. Slides were viewed with a Nihon fluorescent microscope using Q.E.D. software.

[0105] D. Coimmunoprecipitation Assays.

[0106] Nuclear extracts from mock treated U20S cells or cells exposed to 1 or 8 Gy gamma irradiation were prepared as described by Waterman et al, 1998 Nature Genet., 19:175-178. Endogenous 53BP1, Mre11, or p95/NBS were immunoprecipitated by binding 1 μg/reaction of monoclonal anti-53BP1, or 1 μg/reaction purified Mre11 or p95/NBS antisera (Calbiochem) in 1× IP buffer [25 mM Hepes (pH 7.4)/100 mM NaCl/5 mM MgCl₂/100 mM EDTA/0.2 mg/ml BSA/0.1% Tween-20] to protein-G sepharose beads (Amersham) for 1 hour. After washing three times with 1× IP buffer, 100 μg nuclear extract was added and proteins allowed to bind for 1 hour at 4° C. The beads were washed and the proteins resolved on 6% SDS-PAGE, transferred to PVDF membrane and were visualized by western blot using the antibodies described in Part A above.

EXAMPLE 2 53Bp1 Forms Nuclear Foci in Response to Ionizing Radiation

[0107] To investigate whether 53Bp1 participates in the cellular responses to DNA damage, the monoclonal antibodies of Example 1, Part A, directed against the carboxy terminus of 53Bp1 were used in an immunoblot analysis of whole cell extracts (WCE) from the transiently transfected U2OS cells ectopically expressing full length HA-tagged 53Bp1, HA53Bp1ΔBRCT, and HA53Bp1C312. These antibodies recognized specifically 53Bp1 as shown by immunoblot analysis, i.e., the antibodies bound to HA53Bp1 and HA53Bp1C312, but not HA53Bp1ΔBRCT.

[0108] Immunoblotting of endogenous 53Bp1 using nuclear extracts from cells either mock treated or exposed to 8 Gy ionizing radiation indicate that the steady state levels of 53Bp1 remain unchanged after gamma irradiation and that 53Bp1 is not detectably modified in response to DNA damage. Although the predicted molecular mass of 53Bp1 is approximately 217 kDa, the protein migrates significantly slower than its predicted size which is consistent with previous reports [Iwabuchi (1998) cited above].

[0109] Immunofluorescence experiments indicate that 53Bp1 is a uniformly distributed protein in the nuclei of unirradiated cells. However, following 8 Gy gamma irradiation, the subcellular distribution of 53Bp1 is altered to form distinct nuclear foci (i.e., ionizing radiation-induced foci or IRIF) in U20S cells. Nuclear foci formation is not limited to U20S cells, as 53Bp1 also relocalizes to foci in DLD1 and a variety of other normal cells under the same conditions (see below).

EXAMPLE 3 53Bp1 Localizes to Sites of DNA Double Stranded Breaks

[0110] To determine the type of DNA damage to which 53Bp1 was responding, cells were treated with either ionizing radiation, etoposide, hydroxyurea (HU), or ultraviolet (UV) irradiation and stained for 53Bp1, as described in Example 1.

[0111] 53Bp1-containing foci are found specifically in cells exposed to ionizing radiation and etoposide, which cause DNA DSBs, and do not form in the nuclei of cells exposed to 50J/m² UV light and stained 1-6 hours later, or in cells exposed to HU for 1 to 25 hours. These data show that 53Bp1 is relocalizing to DNA DSBs specifically.

[0112] In response to the agents that induce DNA double-strand breaks (DSBs), 53Bp1 localized at discrete foci within the nucleus of interphase cells. These foci appeared within 5 minutes after exposure to ionizing radiation.

EXAMPLE 4 53Bp1 Nuclear Foci Form in a Dose and Time Dependent Manner

[0113] Based on the response of 53Bp1 to ionizing radiation, experiments were performed to investigate whether this response was dose and time dependent. Ionizing radiation induces DNA DSBs at a linear rate of approximately 25 DSBs per human cell per Gy [rather than 36, as indicated by Lobrich (1995), cited above].

[0114] A. Dose Dependence

[0115] U2OS cells were exposed to 0.5, 1, 2, 4, and 8 Gy and these cells were stained one hour post-irradiation. The dose dependence of 53Bp1 foci formation in these cells was observed.

[0116] 53Bp1-containing foci appeared after exposure of cells to 0.5 Gy and increased numbers were formed with increasing irradiation dose (see FIG. 1A). Moreover, the number of foci increase almost linearly with increasing irradiation dose and hence the number of DSB per cell.

[0117] B. Time Dependence

[0118] The time course for 53Bp1 foci formation following exposure to ionizing radiation was determined by exposing growing U2OS cells to 1 Gy of ionizing radiation and fixing the cells at 5, 15, 30, 45 and 60 minutes and 2, 4, 6, 8, 12, and 16 hours post-irradiation.

[0119] Ionizing radiation-induced foci appeared within 15 minutes and greater than 90% of the nuclei contained 53Bp1 foci (see FIGS. 1B and 1C). The number of foci peak at 30 minutes and decrease to baseline values by 16 to 24 hours. These data are consistent with the hypothesis that 53Bp1 localizes to DNA DSBs and is removed when repair is complete.

[0120] In summary, the number of foci was proportional to the dose of ionizing radiation, peaked at the 30 minute time point and then decreased with biphasic kinetics that exhibited fast and slow components.

EXAMPLE 5 53Bp1 Colocalizes With, But Does Not Interact With, the MRE11/RAD50/NBS Complex

[0121] The Mre11, Rad50 and NBS/p95 complex localizes to DNA DSBs in the form of ionizing radiation-induced foci [Maser (1997) and Nelms (1998), both cited above] which look similar to the 53Bp1-containing ionizing radiation-induced foci. Although 53Bp1-containing nuclear foci appear within 15 minutes of 1 Gy ionizing radiation, Mre11 and NBS foci appear four to eight hours post-irradiation and are visualized best following exposure to between 8 and 12 Gy.

[0122] A. 53Bp1 Colocalizes With the Mre11/Rad50/NBS/p95 Complex

[0123] To investigate further the site of 53Bp1 relocalization in the cellular response to DNA damage, an experiment was performed to determine whether 53Bp1 colocalizes with these proteins and with the promyelocytic leukemia protein (PML), another protein that forms nuclear foci as part of the nuclear domain 10 (ND10) structure [Ishov, A. M. et al, 1999 J. Cell. Biol., 147:221-234], both in the absence and presence of DNA damage. U2OS cells were fixed 8 hours after exposure to 8 Gy irradiation and were stained with antibodies recognizing 53Bp1, Mre11, NBS and/or PML.

[0124] The 53Bp1 foci colocalize with Mre11 and NBS ionizing radiation-induced foci, i.e., with the Mre11/Rad50/NBS complex. There was no colocalization of 53Bp1 and the control protein, PML. These data indicate that the 53Bp1 and Mre11/NBS colocalization is specific.

[0125] B. 53Bp1 Does Not Interact With the Mre11/Rad50/NBS Complex

[0126] To determine if 53Bp1 was also part of this complex, a coprecipitation assay was performed as described in Example 1, Part D. Although Mre11, Rad50 and NBS could be co-immunoprecipitated under a variety of conditions, 53Bp1 never co-precipitated with this complex. These data do not support an interaction between these proteins under these conditions. However, these data do not preclude a functional interaction among the proteins as suggested by the colocalization data.

EXAMPLE 6 53Bp1 Foci Formation is Altered With Wortmannin Treatment

[0127] As an additional method to confirm that 53Bp1 plays a role in the DNA repair pathway, the transiently transfected U2OS cells of Example 1 were exposed to the fungal metabolite wortmannin and stained for 53Bp1 at various time points following 1 Gy ionizing radiation.

[0128] Although 53Bp1 ionizing radiation-induced foci were present in some cells, the kinetics of foci formation were significantly altered. When compared to untreated controls, the wortmannin treated cells contained much lower numbers of foci at 30 minutes and the foci numbers did not decrease significantly over time (FIGS. 2A and 2B). Moreover, the percentage of cells containing 53Bp1 ionizing radiation-induced foci was reduced almost 50% at 30 minutes by the presence of wortmannin and then remained elevated over time relative to the control.

[0129] These data indicate that 53Bp1 localization is partially inhibited when DNA-PK, and hence DNA DSB repair, is inhibited by wortmannin. Wortmannin, which slows repair of DNA double-strand breaks (DSBs) slowed both the appearance and resolution of the 53Bp1 foci. These data provide further support that 53Bp1 plays a role in the response of cells to DNA damage.

[0130] In a similar study using caffeine rather than wortmannin, caffeine, which abrogates the DNA damage cell cycle checkpoint [Lau and Pardee 1982 Proc. Natl. Acad. Sci., USA, 79:2942-2946] did not affect 53Bp1 focus formation or dispersion in response to ionizing radiation. DNA-PK is theorized to be the kinase responsible for 53Bp1 relocalization because caffeine, which inhibits ATM and ATR, but not DNA-PK, did not affect 53Bp1 focus formation or dispersion.

EXAMPLE 7 53Bp1 Ionizing Radiation-Induced Foci Formation Does Not Require ATM or NIBRIN

[0131] Because 53Bp1 participates in the cellular responses to DNA damage, this experiment investigated whether 53Bp1 foci formation was dependent on ATM or Nibrin (NBS/p95).

[0132] A. ATM Experiments

[0133] 53Bp1 foci formation was examined in primary AT-1BR and AT-5BI cells which have mutations in the ATM gene and are derived from patients with ataxia telangiectasia. These AT mutated cells are deficient in their ability to signal the presence of DNA damage and are acutely sensitive to ionizing radiation. The ATM protein is activated following DNA damage as part of the DNA damage checkpoint. ATM is important for cell cycle arrest and appears to be needed for repair of chromosomal damage as well [Cornforth, M. N., and Bedford, J. S. 1985 Science, 227:1589-1591; Murnane, J. P. 1995 Cancer Metastasis Rev.,14:17-29 and 14(3):253-4; Pandita, T. K., and Hittelman, W. N. 1992 Radiat. Res., 130: 94-103].

[0134] The response in primary AT fibroblasts was compared to the normal counterpart cell line AG1522 after exposure to ionizing radiation. When exposed to 1 Gy of ionizing radiation and stained 15 minutes post-irradiation there was no significant difference in the cells' ability to form 53Bp1-containing nuclear foci when the AT mutated cells are compared to the AG1522 cells. These data indicate that relocalization of 53Bp1 in response to ionizing radiation can occur in the absence of ATM.

[0135] B. Nibrin Experiments

[0136] To determine if nibrin (NBS/p95) is required for formation of 53Bp1 ionizing radiation-induced foci, primary fibroblasts from patients with Nijmegen Breakage Syndrome were studied. The NBS cell lines 780816 and 880823 contain truncated versions of NBS and are radiosensitive, have radioresistant DNA synthesis and elevated levels of chromosomal aberrations [van der Burgt, I. et al, 1996 J. Med. Genet., 33:153-156]. These NBS mutated cells were irradiated with 1 Gy, fixed, and stained with anti-53Bp1 15 minutes post-irradiation.

[0137] Many nuclear foci similar to those observed in irradiated U20S cells were observed in the NBS mutated cells, so treated. These data suggest that the formation of 53Bp1 ionizing radiation-induced foci does not require nibrin.

EXAMPLE 8 53Bp1 Ionizing Radiation-Induced Foci Are Present in Tumor Cell Lines in the Absence of Exogenous DNA Damage

[0138] One cause of chromosomal instability, a hallmark of cancer cells, may stem from defects in DNA repair and DNA damage checkpoint genes leading to accumulations of DNA DSBs and increased chromosomal abnormalities. To monitor the presence of DNA DSBs, and to determine if there is an increased number of double stranded breaks in cancer cells, the tumor cell lines and normal primary cells identified specifically in Table 1 below, were incubated with the anti-53Bp1 antibody and examined by immunofluorescent microscopy as described in Example 1.

[0139] Table 1 provides an indication of whether such cell lines exhibit a low or high number of 53Bp1 foci in the absence of exposure to ionizing radiation. In the absence of exposure to DNA damaging agents, 53Bp1 foci were not evident in primary cells (normal fibroblast or osteoblast cells), but were evident in cancer cell lines. A high number of 53Bp1 foci were present in cells expected to have DNA damage lesions, such as MO59J, which lack DNA-PK, and HCC1937 cells, which lack functional BRCA1. The cancer cell lines SW480 and HCT11C also contained highly elevated numbers of 53Bp1 foci in the absence of DNA damage. Other tumor cell lines, also had a high number of 53Bp1 foci in the absence of exposure to DNA damaging agents. TABLE 1 Tumor Cell Lines Number of 53Bp1 Foci HCC1937 Very High (>20 per cell) HCTl16 High (>10 per cell) SW480 High (>10 per cell) MO59J High (>10 per cell) HT29 Low (˜1 per cell) MCF7 Low (˜1 per cell) U2OS Low (˜1 per cell) Normal Primary Cells Normal Dermal Fibroblasts Low (<1 per cell) Normal Osteoblasts Low (<1 per cell)

[0140] These data suggest that many tumor cells contain DNA DSBs in the absence of exogenous DNA damaging agents. As expected, the number of 53Bp1 foci were elevated in cells defective for the DNA repair proteins BRCA1 and DNA-PK (HCC1937 and M059J, respectively) without exposure to DNA damaging agents. This is further indication that it is DNA DSBs to which 53Bp1 is localizing. The fact that 53Bp1 foci were observed in some cancer cells, but not in primary cells, suggests that these cancer cell lines contain DNA DSBs, which may be the cause of chromosomal instability so common in tumors. These data suggest that creating molecules that block 53Bp1 provides a novel cancer therapeutic agent.

EXAMPLE 9 Mapping the Region of 53Bp1 That is Required for Its Ability to Form Ionizing Radiation/Induced Foci

[0141] The following experiments were performed to identify the minimal focus-targeting (FT) domain of the relatively large (1972 amino acids) 53Bp1 protein [SEQ ID NO: 2]. The FT domain is the portion of the protein sequence required for targeting to ionizing radiation-induced foci. The FT domain participates in the mechanism of 53Bp1 relocalization. In addition, fragments of 53Bp1 that retain the ability to form ionizing radiation-induced foci act as dominant negative mutants by competing with endogenous 53Bp1 for localization to the IR-induced 53Bp1-binding sites.

[0142] There are two identifiable structural motifs in 53Bp1: a nuclear localization signal (NLS) between amino acids residues 1668-1685 of SEQ ID NO: 2 and two BRCT domains between amino acid residues 1724-1964 of SEQ ID NO: 2. The rest of the protein shows no obvious homology to any other protein.

[0143] A series of 53Bp1 deletion mutants was generated and expressed as HA-tagged proteins in U2OS cells. These cells were examined by immuno-fluorescence for their intracellular localization in response to ionizing radiation, as described in Examples 1 and 2. Nuclear localization is considered to be a prerequisite for targeting of 53Bp1 to ionizing radiation-induced foci. Therefore, all the mutants were constructed so that they retained the NLS of 53Bp1.

[0144] The results of this analysis are reported in Table 2 and show that three quarters of the 53Bp1 sequence, including the BRCT domains, are dispensable for relocalization in response to ionizing radiation. Specifically, amino acid residues 1220-1711 of SEQ ID NO: 2 are sufficient for the ability of 53Bp1 to form ionizing radiation-induced foci. TABLE 2 Ionizing Radiation- 53Bp1 Protein/Fragment Induced Foci Localization full-length (AA residues 1-1972 Yes SEQ ID NO: 2) AA residues 1-1711 (deletion of AA Yes residues 1712-1972) of SEQ ID NO: 2 AA residues 1661-1972 (deletion of No residues 1-1660) of SEQ ID NO: 2 AA residues 1-1053 and 1220-1972 (dele- Yes tion of residues 1054-1219) of SEQ ID NO: 2 AA residues 1-1053 and 1411-1972 No (deletion of residues 1054-1410) of SEQ ID NO: 2 AA residues 1-34 and 1047-1972 (dele- Yes tion of residues 35-1046) of SEQ ID NO: 2 AA residues 1-34 and 1047-1711 (dele- Yes tion of residues 35-1046 and 1712-1972) of SEQ ID NO: 2 AA residues 1-34 and 1220-1711 (dele- Yes tion of residues 35-1219 and 1712-1972) of SEQ ID NO: 2 AA residues 1-34 and 1411-1711 (dele- No tion of residues 35-1410 and 1712-1972) of SEQ ID NO: 2

[0145] As demonstrated by the above Examples 1-8, within five minutes of cellular exposure to DNA DSB-inducing agents, 53Bp1 relocalizes from a homogenous nuclear distribution to very distinct nuclear foci at sites of DNA DSBs. First, 53Bp1 ionizing radiation-induced foci are seen when cells are treated with agents that induce DNA DSBs including gamma irradiation and etoposide. The ionizing radiation-induced foci are not present when cells are treated with HU, causing a replication block, or UV light, which causes the formation of pyrimidine dimers. Secondly, there is a distinct correlation between the number of 53Bp1 foci formed per Gy and the number of DNA DSBs per Gy. This suggests that the number of 53Bp1 foci approximate the number of DNA DSBs. Additionally, the kinetics of DNA DSB repair correlates with the kinetics of resolution of 53Bp1 ionizing radiation-induced foci over time. Thirdly, 53Bp1 ionizing radiation-induced foci colocalize with Mre11 and NBS ionizing radiation-induced foci which have been shown to localize to DNA DSBs. Lastly, the rate of 53Bp1 ionizing radiation-induced foci formation is significantly altered by wortmannin, a drug known to decrease the rate of DNA repair by inhibiting DNA-PK [Boulton, S. et al, 1996 Carcinogenesis, 17:2285-2290]. Taken together, these data provide strong evidence that 53Bp1 relocalizes to sites of DNA DSBs and remains localized until repair is complete.

[0146] Although other known proteins, identified above in the background section, either form nuclear foci or redistribute the foci in response to DNA damaging agents, none respond to DNA breaks by relocalizing to form foci within 5 minutes similar to 53Bp1. Moreover, the number of foci per cell is not proportional to the number of DNA DSBs and the fraction of cells containing foci for many of the proteins is low relative to the data for 53Bp1.

[0147] 53Bp1 is part of the DNA damage checkpoint or repair pathways. 53Bp1 relocalizes to DNA DSBs even in cells that lack ATM, NBS, or BRCA1. These data indicate that these proteins are not required for the relocalization of 53Bp1 following irradiation. However, the rate of relocalization is likely affected. At five minutes post-irradiation, cells contain an increased number of foci, although the number of foci peak between 15 and 30 minutes. The fast, although not immediate, response implies that 53Bp1 is not the earliest cellular response after DNA damage. The DNA damage checkpoint kinase, ATM, is activated within two to five minutes [Banin, S. et al, 1998 Science, 281:1674-1677]. Ku is known to bind DNA ends quickly followed by recruitment of DNA-PK [Smith, G. C., and Jackson, S. P. 1999 Genes Dev., 13:916-934]. However, the time course of 53Bp1 ionizing radiation-induced foci localization implies an active process of recruitment of 53Bp1 to the site of DNA DSBs. The active recruitment of 53Bp1 is also supported by the data that wortmannin inhibits this recruitment.

[0148] 53Bp1 has homology to the budding yeast Rad9, which is required for activation of the DNA damage checkpoint and is now proposed to participate in DNA repair through the NHEJ pathway [de la Torre-Ruiz, M., and Lowndes, N. F. 2000 FEBS Lett., 467:311-315; Weinert and Hartwell, 1988]. It is theorized that 53Bp1 has a similar role in mammalian cells. Because 53Bp1 responds early to DNA damage, it is likely a sensor protein which is necessary for proper signaling in either the checkpoint or repair pathways. ATM is activated within minutes following exposure to DNA double strand break agents. While the peak of 53Bp1 foci occurs at 15-30 minutes after DNA damage, foci are detected within five minutes of ionizing radiation. Thus, the initial response of 53Bp1 is fast enough to be consistent with 53Bp1 being part of the ATM-Chk2 DNA damage checkpoint pathway. Alternatively, or in addition, 53Bp1 is part of the NHEJ pathway as suggested by the colocalization with Mre11/Rad50/NBS. The fact that wortmannin, an inhibitor of PI-3-like kinases, decreases the rate of 53Bp1 recruitment to sites of DNA breaks suggests that DNA-PK or ATM is involved. 53Bp1 functions in the DNA damage checkpoint or repair pathway.

[0149] 53Bp1 binds to the p53 DNA binding domain and not the transactivation domain, as a transactivator would. No p53 was detected coimmunoprecipitating with 53Bp1, however this does not preclude their potential interaction in vivo since p53 also colocalizes with 53Bp1 and it may not be possible to extract 53Bp1/p53 complexes from these foci.

[0150] Taken together, these data of Examples 1-9 indicate that 53Bp1 localizes to sites of DNA breaks earlier than other detectable proteins and colocalizes with other proteins that function in DNA repair and the DNA damage checkpoint. The recruitment of 53Bp1 to sites of DNA damage supports a role in DNA DSB processing and/or DNA damage signaling. In support of a role in DNA damage signaling, 53Bp1 has the highest similarity of all human sequences in the public databases to the C. elegans T05F1 open reading frame, which in turn is the C. elegans sequence with the highest similarity to the S. cerevisiae Rad9p and S. pombe Crb2p/Rhp9p sequences. Recruitment of 53Bp1 to sites of DNA damage represents an important step for DNA repair and/or DNA damage checkpoint control.

[0151] The documents and publications cited above are incorporated herein by reference. Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

1 2 1 6266 DNA human 53Bp1 CDS (174)..(6089) 1 cgttgtttgg cgtgtttttt tttttgtttt ttgtcactgc ctgcctgggt cctgcccgag 60 gtctccatcc tcggtttccc tgtccttgcc ccgggccctg ggagtgctct ggaaggctgc 120 gcagtattgg aggggacaga atgaccttcc ggccttgagt ccctggggag cag atg 176 Met 1 gac cct act gga agt cag ttg gat tca gat ttc tct cag caa gat act 224 Asp Pro Thr Gly Ser Gln Leu Asp Ser Asp Phe Ser Gln Gln Asp Thr 5 10 15 cct tgc ctg ata att gaa gat tct cag cct gaa agc cag gtt cta gag 272 Pro Cys Leu Ile Ile Glu Asp Ser Gln Pro Glu Ser Gln Val Leu Glu 20 25 30 gat gat tct ggt tct cac ttc agt atg cta tct cga cac ctt cct aat 320 Asp Asp Ser Gly Ser His Phe Ser Met Leu Ser Arg His Leu Pro Asn 35 40 45 ctc cag acg cac aaa gaa aat cct gtg ttg gat gtt gtg tcc aat cct 368 Leu Gln Thr His Lys Glu Asn Pro Val Leu Asp Val Val Ser Asn Pro 50 55 60 65 gaa caa aca gct gga gaa gaa cga gga gac ggt aat agt ggg ttc aat 416 Glu Gln Thr Ala Gly Glu Glu Arg Gly Asp Gly Asn Ser Gly Phe Asn 70 75 80 gaa cat ttg aaa gaa aac aag gtt gca gac cct gtg gat tct tct aac 464 Glu His Leu Lys Glu Asn Lys Val Ala Asp Pro Val Asp Ser Ser Asn 85 90 95 ttg gac aca tgt ggt tcc atc agt cag gtc att gag cag tta cct cag 512 Leu Asp Thr Cys Gly Ser Ile Ser Gln Val Ile Glu Gln Leu Pro Gln 100 105 110 cca aac agg aca agc agt gtt ctg gga atg tca gtg gaa tct gct cct 560 Pro Asn Arg Thr Ser Ser Val Leu Gly Met Ser Val Glu Ser Ala Pro 115 120 125 gct gtg gag gaa gag aag gga gaa gag ttg gaa cag aag gag aaa gag 608 Ala Val Glu Glu Glu Lys Gly Glu Glu Leu Glu Gln Lys Glu Lys Glu 130 135 140 145 aag gaa gaa gat act tca ggc aat act aca cat tcc ctt ggt gct gaa 656 Lys Glu Glu Asp Thr Ser Gly Asn Thr Thr His Ser Leu Gly Ala Glu 150 155 160 gat act gcc tca tca cag ttg ggt ttt ggg gtt ctg gaa ctc tcc cag 704 Asp Thr Ala Ser Ser Gln Leu Gly Phe Gly Val Leu Glu Leu Ser Gln 165 170 175 agc cag gat gtt gag gaa aat act gtg cca tat gaa gtg gac aaa gag 752 Ser Gln Asp Val Glu Glu Asn Thr Val Pro Tyr Glu Val Asp Lys Glu 180 185 190 cag cta caa tca gta acc acc aac tct ggt tat acc agg ctg tct gat 800 Gln Leu Gln Ser Val Thr Thr Asn Ser Gly Tyr Thr Arg Leu Ser Asp 195 200 205 gtg gat gct aat act gca att aag cat gaa gaa cag tcc aac gaa gat 848 Val Asp Ala Asn Thr Ala Ile Lys His Glu Glu Gln Ser Asn Glu Asp 210 215 220 225 atc ccc ata gca gaa cag tcc agc aag gac atc cct gtg aca gca cag 896 Ile Pro Ile Ala Glu Gln Ser Ser Lys Asp Ile Pro Val Thr Ala Gln 230 235 240 ccc agt aag gat gta cat gtt gta aaa gag caa aat cca cca cct gca 944 Pro Ser Lys Asp Val His Val Val Lys Glu Gln Asn Pro Pro Pro Ala 245 250 255 agg tca gag gac atg cct ttt agc ccc aaa gca tct gtt gct gct atg 992 Arg Ser Glu Asp Met Pro Phe Ser Pro Lys Ala Ser Val Ala Ala Met 260 265 270 gaa gca aaa gaa cag ttg tct gca caa gaa ctt atg gaa agt gga ctg 1040 Glu Ala Lys Glu Gln Leu Ser Ala Gln Glu Leu Met Glu Ser Gly Leu 275 280 285 cag att cag aag tca cca gag cct gag gtt ttg tca act cag gaa gac 1088 Gln Ile Gln Lys Ser Pro Glu Pro Glu Val Leu Ser Thr Gln Glu Asp 290 295 300 305 ttg ttt gac cag agc aat aaa aca gta tct tct gat ggt tgc tct act 1136 Leu Phe Asp Gln Ser Asn Lys Thr Val Ser Ser Asp Gly Cys Ser Thr 310 315 320 cct tca agg gag gaa ggt ggg tgt tct ttg gct tcc act cct gcc acc 1184 Pro Ser Arg Glu Glu Gly Gly Cys Ser Leu Ala Ser Thr Pro Ala Thr 325 330 335 act ctg cat ctc ctg cag ctc tct ggt cag agg tcc ctt gtt cag gac 1232 Thr Leu His Leu Leu Gln Leu Ser Gly Gln Arg Ser Leu Val Gln Asp 340 345 350 agt ctt tcc acg aat tct tca gat ctt gtt gct cct tct cct gat gct 1280 Ser Leu Ser Thr Asn Ser Ser Asp Leu Val Ala Pro Ser Pro Asp Ala 355 360 365 ttc cga tct act cct ttt atc gtt cct agc agt ccc aca gag caa gaa 1328 Phe Arg Ser Thr Pro Phe Ile Val Pro Ser Ser Pro Thr Glu Gln Glu 370 375 380 385 ggg aga caa gat aag cca atg gac acg tca gtg tta tct gaa gaa gga 1376 Gly Arg Gln Asp Lys Pro Met Asp Thr Ser Val Leu Ser Glu Glu Gly 390 395 400 gga gag cct ttt cag aag aaa ctt caa agt ggt gaa cca gtg gag tta 1424 Gly Glu Pro Phe Gln Lys Lys Leu Gln Ser Gly Glu Pro Val Glu Leu 405 410 415 gaa aac ccc cct ctc ctg cct gag tcc act gta tca cca caa gcc tca 1472 Glu Asn Pro Pro Leu Leu Pro Glu Ser Thr Val Ser Pro Gln Ala Ser 420 425 430 aca cca ata tct cag agc aca cca gtc ttc cct cct ggg tca ctt cct 1520 Thr Pro Ile Ser Gln Ser Thr Pro Val Phe Pro Pro Gly Ser Leu Pro 435 440 445 atc cca tcc cag cct cag ttt tct cat gac att ttt att cct tcc cca 1568 Ile Pro Ser Gln Pro Gln Phe Ser His Asp Ile Phe Ile Pro Ser Pro 450 455 460 465 agt ctg gaa gaa caa tca aat gat ggg aag aaa gat gga gat atg cat 1616 Ser Leu Glu Glu Gln Ser Asn Asp Gly Lys Lys Asp Gly Asp Met His 470 475 480 agt tca tct ttg aca gtt gag tgt tct aaa act tca gag att gaa cca 1664 Ser Ser Ser Leu Thr Val Glu Cys Ser Lys Thr Ser Glu Ile Glu Pro 485 490 495 aag aat tcc cct gag gat ctt ggg cta tct ttg aca ggg gat tct tgc 1712 Lys Asn Ser Pro Glu Asp Leu Gly Leu Ser Leu Thr Gly Asp Ser Cys 500 505 510 aag ttg atg ctt tct aca agt gaa tat agt cag tcc cca aag atg gag 1760 Lys Leu Met Leu Ser Thr Ser Glu Tyr Ser Gln Ser Pro Lys Met Glu 515 520 525 agc ttg agt tct cac aga att gat gaa gat gga gaa aac aca cag att 1808 Ser Leu Ser Ser His Arg Ile Asp Glu Asp Gly Glu Asn Thr Gln Ile 530 535 540 545 gag gat acg gaa ccc atg tct cca gtt ctc aat tct aaa ttt gtt cct 1856 Glu Asp Thr Glu Pro Met Ser Pro Val Leu Asn Ser Lys Phe Val Pro 550 555 560 gct gaa aat gat agt atc ctg atg aat cca gca cag gat ggt gaa gta 1904 Ala Glu Asn Asp Ser Ile Leu Met Asn Pro Ala Gln Asp Gly Glu Val 565 570 575 caa ctg agt cag aat gat gac aaa aca aag gga gat gat aca gac acc 1952 Gln Leu Ser Gln Asn Asp Asp Lys Thr Lys Gly Asp Asp Thr Asp Thr 580 585 590 agg gat gac att agt att tta gcc act ggt tgc aag ggc aga gaa gaa 2000 Arg Asp Asp Ile Ser Ile Leu Ala Thr Gly Cys Lys Gly Arg Glu Glu 595 600 605 acg gta gca gaa gat gtt tgt att gat ctc act tgt gat tcg ggg agt 2048 Thr Val Ala Glu Asp Val Cys Ile Asp Leu Thr Cys Asp Ser Gly Ser 610 615 620 625 cag gca gtt ccg tca cca gct act cga tct gag gca ctt tct agt gtg 2096 Gln Ala Val Pro Ser Pro Ala Thr Arg Ser Glu Ala Leu Ser Ser Val 630 635 640 tta gat cag gag gaa gct atg gaa att aaa gaa cac cat cca gag gag 2144 Leu Asp Gln Glu Glu Ala Met Glu Ile Lys Glu His His Pro Glu Glu 645 650 655 ggg tct tca ggg tct gag gtg gaa gaa atc cct gag aca cct tgt gaa 2192 Gly Ser Ser Gly Ser Glu Val Glu Glu Ile Pro Glu Thr Pro Cys Glu 660 665 670 agt caa gga gag gaa ctc aaa gaa gaa aat atg gag agt gtt ccg ttg 2240 Ser Gln Gly Glu Glu Leu Lys Glu Glu Asn Met Glu Ser Val Pro Leu 675 680 685 cac ctt tct ctg act gaa act cag tcc caa ggg ttg tgt ctt caa aag 2288 His Leu Ser Leu Thr Glu Thr Gln Ser Gln Gly Leu Cys Leu Gln Lys 690 695 700 705 gaa atg cca aaa aaa gaa tgc tca gaa gct atg gaa gtt gaa acc agt 2336 Glu Met Pro Lys Lys Glu Cys Ser Glu Ala Met Glu Val Glu Thr Ser 710 715 720 gtg att agt att gat tcc cct caa aag ttg gca ata ctt gac caa gaa 2384 Val Ile Ser Ile Asp Ser Pro Gln Lys Leu Ala Ile Leu Asp Gln Glu 725 730 735 ttg gaa cat aag gaa cag gaa gct tgg gaa gaa gct act tca gag gac 2432 Leu Glu His Lys Glu Gln Glu Ala Trp Glu Glu Ala Thr Ser Glu Asp 740 745 750 tcc agt gtt gtc att gta gat gtg aaa gag cca tct ccc aga gtt gat 2480 Ser Ser Val Val Ile Val Asp Val Lys Glu Pro Ser Pro Arg Val Asp 755 760 765 gtt tct tgt gaa cct ttg gag gga gtg gag aag tgc tca gat tcc cag 2528 Val Ser Cys Glu Pro Leu Glu Gly Val Glu Lys Cys Ser Asp Ser Gln 770 775 780 785 tca tgg gag gat att gct cca gaa ata gaa cca tgt gct gag aat aga 2576 Ser Trp Glu Asp Ile Ala Pro Glu Ile Glu Pro Cys Ala Glu Asn Arg 790 795 800 tta gac acc aag gaa gaa aag agt gta gaa tat gaa gga gat ctg aaa 2624 Leu Asp Thr Lys Glu Glu Lys Ser Val Glu Tyr Glu Gly Asp Leu Lys 805 810 815 tca ggg act gca gaa aca gaa cct gta gag caa gat tct tca cag cct 2672 Ser Gly Thr Ala Glu Thr Glu Pro Val Glu Gln Asp Ser Ser Gln Pro 820 825 830 tcc tta cct tta gtg aga gca gat gat cct tta aga ctt gac cag gag 2720 Ser Leu Pro Leu Val Arg Ala Asp Asp Pro Leu Arg Leu Asp Gln Glu 835 840 845 ttg cag cag ccc caa act cag gag aaa aca agt aat tca tta aca gaa 2768 Leu Gln Gln Pro Gln Thr Gln Glu Lys Thr Ser Asn Ser Leu Thr Glu 850 855 860 865 gac tca aaa atg gct aat gca aag cag cta agc tca gat gca gag gcc 2816 Asp Ser Lys Met Ala Asn Ala Lys Gln Leu Ser Ser Asp Ala Glu Ala 870 875 880 cag aag ctg ggg aag ccc tct gcc cat gcc tca caa agc ttc tgt gaa 2864 Gln Lys Leu Gly Lys Pro Ser Ala His Ala Ser Gln Ser Phe Cys Glu 885 890 895 agt tct agt gaa acc cca ttt cat ttc act ttg cct aaa gaa ggt gat 2912 Ser Ser Ser Glu Thr Pro Phe His Phe Thr Leu Pro Lys Glu Gly Asp 900 905 910 atc atc cca cca ttg act ggt gca acc cca cct ctt att ggg cac cta 2960 Ile Ile Pro Pro Leu Thr Gly Ala Thr Pro Pro Leu Ile Gly His Leu 915 920 925 aaa ttg gag ccc aag aga cac agt act cct att ggt att agc aac tat 3008 Lys Leu Glu Pro Lys Arg His Ser Thr Pro Ile Gly Ile Ser Asn Tyr 930 935 940 945 cca gaa agc acc ata gca acc agt gat gtc atg tct gaa agc atg gtg 3056 Pro Glu Ser Thr Ile Ala Thr Ser Asp Val Met Ser Glu Ser Met Val 950 955 960 gag acc cat gat ccc ata ctt ggg agt gga aaa ggg gat tct ggg gct 3104 Glu Thr His Asp Pro Ile Leu Gly Ser Gly Lys Gly Asp Ser Gly Ala 965 970 975 gcc cca gac gtg gat gat aaa tta tgt cta aga atg aaa ctg gtt agt 3152 Ala Pro Asp Val Asp Asp Lys Leu Cys Leu Arg Met Lys Leu Val Ser 980 985 990 cct gag act gag gcg agt gaa gag tct ttg cag ttc aac ctg gaa aag 3200 Pro Glu Thr Glu Ala Ser Glu Glu Ser Leu Gln Phe Asn Leu Glu Lys 995 1000 1005 cct gca act ggt gaa aga aaa aat gga tct act gct gtt gct gag 3245 Pro Ala Thr Gly Glu Arg Lys Asn Gly Ser Thr Ala Val Ala Glu 1010 1015 1020 tct gtt gcc agt ccc cag aag acc atg tct gtg ttg agc tgt atc 3290 Ser Val Ala Ser Pro Gln Lys Thr Met Ser Val Leu Ser Cys Ile 1025 1030 1035 tgt gaa gcc agg caa gag aat gag gct cga agt gag gat ccc ccc 3335 Cys Glu Ala Arg Gln Glu Asn Glu Ala Arg Ser Glu Asp Pro Pro 1040 1045 1050 acc aca ccc atc agg ggg aac ttg ctc cac ttt cca agt tct caa 3380 Thr Thr Pro Ile Arg Gly Asn Leu Leu His Phe Pro Ser Ser Gln 1055 1060 1065 gga gaa gag gag aaa gaa aaa ttg gag ggt gac cat aca atc agg 3425 Gly Glu Glu Glu Lys Glu Lys Leu Glu Gly Asp His Thr Ile Arg 1070 1075 1080 cag agt caa cag cct atg aag ccc att agt cct gtc aag gac cct 3470 Gln Ser Gln Gln Pro Met Lys Pro Ile Ser Pro Val Lys Asp Pro 1085 1090 1095 gtt tct cct gct tcc cag aag atg gtc ata caa ggg cca tcc agt 3515 Val Ser Pro Ala Ser Gln Lys Met Val Ile Gln Gly Pro Ser Ser 1100 1105 1110 cct caa gga gag gca atg gtg aca gat gtg cta gaa gac cag aaa 3560 Pro Gln Gly Glu Ala Met Val Thr Asp Val Leu Glu Asp Gln Lys 1115 1120 1125 gaa gga cgg agt act aat aag gaa aat cct agt aag gcc ttg att 3605 Glu Gly Arg Ser Thr Asn Lys Glu Asn Pro Ser Lys Ala Leu Ile 1130 1135 1140 gaa agg ccc agc caa aat aac ata gga atc caa acc atg gag tgt 3650 Glu Arg Pro Ser Gln Asn Asn Ile Gly Ile Gln Thr Met Glu Cys 1145 1150 1155 tcc ttg agg gtc cca gaa act gtt tca gca gca acc cag act ata 3695 Ser Leu Arg Val Pro Glu Thr Val Ser Ala Ala Thr Gln Thr Ile 1160 1165 1170 aag aat gtg tgt gag cag ggg acc agt aca gtg gac cag aac ttt 3740 Lys Asn Val Cys Glu Gln Gly Thr Ser Thr Val Asp Gln Asn Phe 1175 1180 1185 gga aag caa gat gcc aca gtt cag act gag agg ggg agt ggt gag 3785 Gly Lys Gln Asp Ala Thr Val Gln Thr Glu Arg Gly Ser Gly Glu 1190 1195 1200 aaa cca gtc agt gct cct ggg gat gat aca gag tcg ctc cat agc 3830 Lys Pro Val Ser Ala Pro Gly Asp Asp Thr Glu Ser Leu His Ser 1205 1210 1215 cag gga gaa gaa gag ttt gat atg cct cag cct cca cat ggc cat 3875 Gln Gly Glu Glu Glu Phe Asp Met Pro Gln Pro Pro His Gly His 1220 1225 1230 gtc tta cat cgt cac atg aga aca atc cgg gaa gta cgc aca ctt 3920 Val Leu His Arg His Met Arg Thr Ile Arg Glu Val Arg Thr Leu 1235 1240 1245 gtc act cgt gtc att aca gat gtg tat tat gtg gat gga aca gaa 3965 Val Thr Arg Val Ile Thr Asp Val Tyr Tyr Val Asp Gly Thr Glu 1250 1255 1260 gta gaa aga aaa gta act gag gag act gaa gag cca att gta gag 4010 Val Glu Arg Lys Val Thr Glu Glu Thr Glu Glu Pro Ile Val Glu 1265 1270 1275 tgt cag gag tgt gaa act gaa gtt tcc cct tca cag act ggg ggc 4055 Cys Gln Glu Cys Glu Thr Glu Val Ser Pro Ser Gln Thr Gly Gly 1280 1285 1290 tcc tca ggt gac ctg ggg gat atc agc tcc ttc tcc tcc aag gca 4100 Ser Ser Gly Asp Leu Gly Asp Ile Ser Ser Phe Ser Ser Lys Ala 1295 1300 1305 tcc agc tta cac cgc aca tca agt ggg aca agt ctc tca gct atg 4145 Ser Ser Leu His Arg Thr Ser Ser Gly Thr Ser Leu Ser Ala Met 1310 1315 1320 cac agc agt gga agc tca ggg aaa gga gcc gga cca ctc aga ggg 4190 His Ser Ser Gly Ser Ser Gly Lys Gly Ala Gly Pro Leu Arg Gly 1325 1330 1335 aaa acc agc ggg aca gaa ccc gca gat ttt gcc tta ccc agc tcc 4235 Lys Thr Ser Gly Thr Glu Pro Ala Asp Phe Ala Leu Pro Ser Ser 1340 1345 1350 cga gga ggc cca gga aaa ctg agt cct aga aaa ggg gtc agt cag 4280 Arg Gly Gly Pro Gly Lys Leu Ser Pro Arg Lys Gly Val Ser Gln 1355 1360 1365 aca ggg acg cca gtg tgt gag gag gat ggt gat gca ggc ctt ggc 4325 Thr Gly Thr Pro Val Cys Glu Glu Asp Gly Asp Ala Gly Leu Gly 1370 1375 1380 atc aga cag gga ggg aag gct cca gtc acg cct cgt ggg cgt ggg 4370 Ile Arg Gln Gly Gly Lys Ala Pro Val Thr Pro Arg Gly Arg Gly 1385 1390 1395 cga agg ggc cgc cca cct tct cgg acc act gga acc aga gaa aca 4415 Arg Arg Gly Arg Pro Pro Ser Arg Thr Thr Gly Thr Arg Glu Thr 1400 1405 1410 gct gtg cct ggc ccc ttg ggc ata gag gac att tca cct aac ttg 4460 Ala Val Pro Gly Pro Leu Gly Ile Glu Asp Ile Ser Pro Asn Leu 1415 1420 1425 tca cca gat gat aaa tcc ttc agc cgt gtc gtg ccc cga gtg cca 4505 Ser Pro Asp Asp Lys Ser Phe Ser Arg Val Val Pro Arg Val Pro 1430 1435 1440 gac tcc acc aga cga aca gat gtg ggt gct ggt gct ttg cgt cgt 4550 Asp Ser Thr Arg Arg Thr Asp Val Gly Ala Gly Ala Leu Arg Arg 1445 1450 1455 agt gac tct cca gaa att cct ttc cag gct gct gct ggc cct tct 4595 Ser Asp Ser Pro Glu Ile Pro Phe Gln Ala Ala Ala Gly Pro Ser 1460 1465 1470 gat ggc tta gat gcc tcc tct cca gga aat agc ttt gta ggg ctc 4640 Asp Gly Leu Asp Ala Ser Ser Pro Gly Asn Ser Phe Val Gly Leu 1475 1480 1485 cgt gtt gta gcc aag tgg tca tcc aat ggc tac ttt tac tct ggg 4685 Arg Val Val Ala Lys Trp Ser Ser Asn Gly Tyr Phe Tyr Ser Gly 1490 1495 1500 aaa atc aca cga gat gtc gga gct ggg aag tat aaa ttg ctc ttt 4730 Lys Ile Thr Arg Asp Val Gly Ala Gly Lys Tyr Lys Leu Leu Phe 1505 1510 1515 gat gat ggg tac gaa tgt gat gtg ttg ggc aaa gac att ctg tta 4775 Asp Asp Gly Tyr Glu Cys Asp Val Leu Gly Lys Asp Ile Leu Leu 1520 1525 1530 tgt gac ccc atc ccg ctg gac act gaa gtg acg gcc ctc tcg gag 4820 Cys Asp Pro Ile Pro Leu Asp Thr Glu Val Thr Ala Leu Ser Glu 1535 1540 1545 gat gag tat ttc agt gca gga gtg gtg aaa gga cat agg aag gag 4865 Asp Glu Tyr Phe Ser Ala Gly Val Val Lys Gly His Arg Lys Glu 1550 1555 1560 tct ggg gaa ctg tac tac agc att gaa aaa gaa ggc caa aga aag 4910 Ser Gly Glu Leu Tyr Tyr Ser Ile Glu Lys Glu Gly Gln Arg Lys 1565 1570 1575 tgg tat aag cga atg gct gtc atc ctg tcc ttg gag caa gga aac 4955 Trp Tyr Lys Arg Met Ala Val Ile Leu Ser Leu Glu Gln Gly Asn 1580 1585 1590 aga ctg aga gag cag tat ggg ctt ggc ccc tat gaa gca gta aca 5000 Arg Leu Arg Glu Gln Tyr Gly Leu Gly Pro Tyr Glu Ala Val Thr 1595 1600 1605 cct ctt aca aag gca gca gat atc agc tta gac aat ttg gtg gaa 5045 Pro Leu Thr Lys Ala Ala Asp Ile Ser Leu Asp Asn Leu Val Glu 1610 1615 1620 ggg aag cgg aaa cgg cgc agt aac gtc agc tcc cca gcc acc cct 5090 Gly Lys Arg Lys Arg Arg Ser Asn Val Ser Ser Pro Ala Thr Pro 1625 1630 1635 act gcc tcc agt agc agc agc aca acc cct acc cga aag atc aca 5135 Thr Ala Ser Ser Ser Ser Ser Thr Thr Pro Thr Arg Lys Ile Thr 1640 1645 1650 gaa agt cct cgt gcc tcc atg gga gtt ctc tca ggc aaa aga aaa 5180 Glu Ser Pro Arg Ala Ser Met Gly Val Leu Ser Gly Lys Arg Lys 1655 1660 1665 ctt atc act tct gaa gag gaa cgg tcc cct gcc aag cga ggt cgc 5225 Leu Ile Thr Ser Glu Glu Glu Arg Ser Pro Ala Lys Arg Gly Arg 1670 1675 1680 aag tct gcc aca gta aaa cct ggt gca gta ggg gca gga gag ttt 5270 Lys Ser Ala Thr Val Lys Pro Gly Ala Val Gly Ala Gly Glu Phe 1685 1690 1695 gtg agc ccc tgt gag agt gga gac aac acc ggt gaa ccc tct gcc 5315 Val Ser Pro Cys Glu Ser Gly Asp Asn Thr Gly Glu Pro Ser Ala 1700 1705 1710 ctg gaa gag cag aga ggg cct ttg cct ctc aac aag acc ttg ttt 5360 Leu Glu Glu Gln Arg Gly Pro Leu Pro Leu Asn Lys Thr Leu Phe 1715 1720 1725 ctg ggc tac gca ttt ctc ctt acc atg gcc aca acc agt gac aag 5405 Leu Gly Tyr Ala Phe Leu Leu Thr Met Ala Thr Thr Ser Asp Lys 1730 1735 1740 ttg gcc agc cgc tcc aaa ctg cca gat ggt cct aca gga agc agt 5450 Leu Ala Ser Arg Ser Lys Leu Pro Asp Gly Pro Thr Gly Ser Ser 1745 1750 1755 gaa gaa gag gag gaa ttt ttg gaa att cct cct ttc aac aag cag 5495 Glu Glu Glu Glu Glu Phe Leu Glu Ile Pro Pro Phe Asn Lys Gln 1760 1765 1770 tat aca gaa tcc cag ctt cga gca gga gct ggc tat atc ctt gaa 5540 Tyr Thr Glu Ser Gln Leu Arg Ala Gly Ala Gly Tyr Ile Leu Glu 1775 1780 1785 gat ttc aat gaa gcc cag tgt aac aca gct tac cag tgt ctt cta 5585 Asp Phe Asn Glu Ala Gln Cys Asn Thr Ala Tyr Gln Cys Leu Leu 1790 1795 1800 att gcg gat cag cat tgt cga acc cgg aag tac ttc ctg tgc ctt 5630 Ile Ala Asp Gln His Cys Arg Thr Arg Lys Tyr Phe Leu Cys Leu 1805 1810 1815 gcc agt ggg att cct tgt gtg tct cat gtc tgg gtc cat gat agt 5675 Ala Ser Gly Ile Pro Cys Val Ser His Val Trp Val His Asp Ser 1820 1825 1830 tgc cat gcc aac cag ctc cag aac tac cgt aat tat ctg ttg cca 5720 Cys His Ala Asn Gln Leu Gln Asn Tyr Arg Asn Tyr Leu Leu Pro 1835 1840 1845 gct ggg tac agc ctt gag gag caa aga att ctg gac tgg caa ccc 5765 Ala Gly Tyr Ser Leu Glu Glu Gln Arg Ile Leu Asp Trp Gln Pro 1850 1855 1860 cgt gaa aat cct ttc cag aat ctg aag gta ctc ttg gta tca gac 5810 Arg Glu Asn Pro Phe Gln Asn Leu Lys Val Leu Leu Val Ser Asp 1865 1870 1875 caa cag cag aac ttc ctg gag ctc tgg tct gag atc ctc atg act 5855 Gln Gln Gln Asn Phe Leu Glu Leu Trp Ser Glu Ile Leu Met Thr 1880 1885 1890 ggt ggt gca gcc tct gtg aag cag cac cat tca agt gcc cat aac 5900 Gly Gly Ala Ala Ser Val Lys Gln His His Ser Ser Ala His Asn 1895 1900 1905 aaa gat att gct tta ggg gta ttt gat gtg gtg gtg acg gac ccc 5945 Lys Asp Ile Ala Leu Gly Val Phe Asp Val Val Val Thr Asp Pro 1910 1915 1920 tca tgc cca gcc tcg gtg ctg aag tgt gct gaa gca ttg cag ctg 5990 Ser Cys Pro Ala Ser Val Leu Lys Cys Ala Glu Ala Leu Gln Leu 1925 1930 1935 cct gtg gtg tca caa gag tgg gtg atc cag tgc ctc att gtt ggg 6035 Pro Val Val Ser Gln Glu Trp Val Ile Gln Cys Leu Ile Val Gly 1940 1945 1950 gag aga att gga ttc aag cag cat cca aaa tat aaa cac gat tat 6080 Glu Arg Ile Gly Phe Lys Gln His Pro Lys Tyr Lys His Asp Tyr 1955 1960 1965 gtt tct cac taaagatact tggtcttact ggttttattc cctgctatcg 6129 Val Ser His 1970 tggagattgt gttttaacca ggttttaaat gtgtcttgtg tgtaactgga ttccttgcat 6189 ggatcttgta tatagtttta tttgctgaac ttttatgata aaataaatgt tgaatctctt 6249 tggttgtagt aactggg 6266 2 1972 PRT human 53Bp1 2 Met Asp Pro Thr Gly Ser Gln Leu Asp Ser Asp Phe Ser Gln Gln Asp 1 5 10 15 Thr Pro Cys Leu Ile Ile Glu Asp Ser Gln Pro Glu Ser Gln Val Leu 20 25 30 Glu Asp Asp Ser Gly Ser His Phe Ser Met Leu Ser Arg His Leu Pro 35 40 45 Asn Leu Gln Thr His Lys Glu Asn Pro Val Leu Asp Val Val Ser Asn 50 55 60 Pro Glu Gln Thr Ala Gly Glu Glu Arg Gly Asp Gly Asn Ser Gly Phe 65 70 75 80 Asn Glu His Leu Lys Glu Asn Lys Val Ala Asp Pro Val Asp Ser Ser 85 90 95 Asn Leu Asp Thr Cys Gly Ser Ile Ser Gln Val Ile Glu Gln Leu Pro 100 105 110 Gln Pro Asn Arg Thr Ser Ser Val Leu Gly Met Ser Val Glu Ser Ala 115 120 125 Pro Ala Val Glu Glu Glu Lys Gly Glu Glu Leu Glu Gln Lys Glu Lys 130 135 140 Glu Lys Glu Glu Asp Thr Ser Gly Asn Thr Thr His Ser Leu Gly Ala 145 150 155 160 Glu Asp Thr Ala Ser Ser Gln Leu Gly Phe Gly Val Leu Glu Leu Ser 165 170 175 Gln Ser Gln Asp Val Glu Glu Asn Thr Val Pro Tyr Glu Val Asp Lys 180 185 190 Glu Gln Leu Gln Ser Val Thr Thr Asn Ser Gly Tyr Thr Arg Leu Ser 195 200 205 Asp Val Asp Ala Asn Thr Ala Ile Lys His Glu Glu Gln Ser Asn Glu 210 215 220 Asp Ile Pro Ile Ala Glu Gln Ser Ser Lys Asp Ile Pro Val Thr Ala 225 230 235 240 Gln Pro Ser Lys Asp Val His Val Val Lys Glu Gln Asn Pro Pro Pro 245 250 255 Ala Arg Ser Glu Asp Met Pro Phe Ser Pro Lys Ala Ser Val Ala Ala 260 265 270 Met Glu Ala Lys Glu Gln Leu Ser Ala Gln Glu Leu Met Glu Ser Gly 275 280 285 Leu Gln Ile Gln Lys Ser Pro Glu Pro Glu Val Leu Ser Thr Gln Glu 290 295 300 Asp Leu Phe Asp Gln Ser Asn Lys Thr Val Ser Ser Asp Gly Cys Ser 305 310 315 320 Thr Pro Ser Arg Glu Glu Gly Gly Cys Ser Leu Ala Ser Thr Pro Ala 325 330 335 Thr Thr Leu His Leu Leu Gln Leu Ser Gly Gln Arg Ser Leu Val Gln 340 345 350 Asp Ser Leu Ser Thr Asn Ser Ser Asp Leu Val Ala Pro Ser Pro Asp 355 360 365 Ala Phe Arg Ser Thr Pro Phe Ile Val Pro Ser Ser Pro Thr Glu Gln 370 375 380 Glu Gly Arg Gln Asp Lys Pro Met Asp Thr Ser Val Leu Ser Glu Glu 385 390 395 400 Gly Gly Glu Pro Phe Gln Lys Lys Leu Gln Ser Gly Glu Pro Val Glu 405 410 415 Leu Glu Asn Pro Pro Leu Leu Pro Glu Ser Thr Val Ser Pro Gln Ala 420 425 430 Ser Thr Pro Ile Ser Gln Ser Thr Pro Val Phe Pro Pro Gly Ser Leu 435 440 445 Pro Ile Pro Ser Gln Pro Gln Phe Ser His Asp Ile Phe Ile Pro Ser 450 455 460 Pro Ser Leu Glu Glu Gln Ser Asn Asp Gly Lys Lys Asp Gly Asp Met 465 470 475 480 His Ser Ser Ser Leu Thr Val Glu Cys Ser Lys Thr Ser Glu Ile Glu 485 490 495 Pro Lys Asn Ser Pro Glu Asp Leu Gly Leu Ser Leu Thr Gly Asp Ser 500 505 510 Cys Lys Leu Met Leu Ser Thr Ser Glu Tyr Ser Gln Ser Pro Lys Met 515 520 525 Glu Ser Leu Ser Ser His Arg Ile Asp Glu Asp Gly Glu Asn Thr Gln 530 535 540 Ile Glu Asp Thr Glu Pro Met Ser Pro Val Leu Asn Ser Lys Phe Val 545 550 555 560 Pro Ala Glu Asn Asp Ser Ile Leu Met Asn Pro Ala Gln Asp Gly Glu 565 570 575 Val Gln Leu Ser Gln Asn Asp Asp Lys Thr Lys Gly Asp Asp Thr Asp 580 585 590 Thr Arg Asp Asp Ile Ser Ile Leu Ala Thr Gly Cys Lys Gly Arg Glu 595 600 605 Glu Thr Val Ala Glu Asp Val Cys Ile Asp Leu Thr Cys Asp Ser Gly 610 615 620 Ser Gln Ala Val Pro Ser Pro Ala Thr Arg Ser Glu Ala Leu Ser Ser 625 630 635 640 Val Leu Asp Gln Glu Glu Ala Met Glu Ile Lys Glu His His Pro Glu 645 650 655 Glu Gly Ser Ser Gly Ser Glu Val Glu Glu Ile Pro Glu Thr Pro Cys 660 665 670 Glu Ser Gln Gly Glu Glu Leu Lys Glu Glu Asn Met Glu Ser Val Pro 675 680 685 Leu His Leu Ser Leu Thr Glu Thr Gln Ser Gln Gly Leu Cys Leu Gln 690 695 700 Lys Glu Met Pro Lys Lys Glu Cys Ser Glu Ala Met Glu Val Glu Thr 705 710 715 720 Ser Val Ile Ser Ile Asp Ser Pro Gln Lys Leu Ala Ile Leu Asp Gln 725 730 735 Glu Leu Glu His Lys Glu Gln Glu Ala Trp Glu Glu Ala Thr Ser Glu 740 745 750 Asp Ser Ser Val Val Ile Val Asp Val Lys Glu Pro Ser Pro Arg Val 755 760 765 Asp Val Ser Cys Glu Pro Leu Glu Gly Val Glu Lys Cys Ser Asp Ser 770 775 780 Gln Ser Trp Glu Asp Ile Ala Pro Glu Ile Glu Pro Cys Ala Glu Asn 785 790 795 800 Arg Leu Asp Thr Lys Glu Glu Lys Ser Val Glu Tyr Glu Gly Asp Leu 805 810 815 Lys Ser Gly Thr Ala Glu Thr Glu Pro Val Glu Gln Asp Ser Ser Gln 820 825 830 Pro Ser Leu Pro Leu Val Arg Ala Asp Asp Pro Leu Arg Leu Asp Gln 835 840 845 Glu Leu Gln Gln Pro Gln Thr Gln Glu Lys Thr Ser Asn Ser Leu Thr 850 855 860 Glu Asp Ser Lys Met Ala Asn Ala Lys Gln Leu Ser Ser Asp Ala Glu 865 870 875 880 Ala Gln Lys Leu Gly Lys Pro Ser Ala His Ala Ser Gln Ser Phe Cys 885 890 895 Glu Ser Ser Ser Glu Thr Pro Phe His Phe Thr Leu Pro Lys Glu Gly 900 905 910 Asp Ile Ile Pro Pro Leu Thr Gly Ala Thr Pro Pro Leu Ile Gly His 915 920 925 Leu Lys Leu Glu Pro Lys Arg His Ser Thr Pro Ile Gly Ile Ser Asn 930 935 940 Tyr Pro Glu Ser Thr Ile Ala Thr Ser Asp Val Met Ser Glu Ser Met 945 950 955 960 Val Glu Thr His Asp Pro Ile Leu Gly Ser Gly Lys Gly Asp Ser Gly 965 970 975 Ala Ala Pro Asp Val Asp Asp Lys Leu Cys Leu Arg Met Lys Leu Val 980 985 990 Ser Pro Glu Thr Glu Ala Ser Glu Glu Ser Leu Gln Phe Asn Leu Glu 995 1000 1005 Lys Pro Ala Thr Gly Glu Arg Lys Asn Gly Ser Thr Ala Val Ala 1010 1015 1020 Glu Ser Val Ala Ser Pro Gln Lys Thr Met Ser Val Leu Ser Cys 1025 1030 1035 Ile Cys Glu Ala Arg Gln Glu Asn Glu Ala Arg Ser Glu Asp Pro 1040 1045 1050 Pro Thr Thr Pro Ile Arg Gly Asn Leu Leu His Phe Pro Ser Ser 1055 1060 1065 Gln Gly Glu Glu Glu Lys Glu Lys Leu Glu Gly Asp His Thr Ile 1070 1075 1080 Arg Gln Ser Gln Gln Pro Met Lys Pro Ile Ser Pro Val Lys Asp 1085 1090 1095 Pro Val Ser Pro Ala Ser Gln Lys Met Val Ile Gln Gly Pro Ser 1100 1105 1110 Ser Pro Gln Gly Glu Ala Met Val Thr Asp Val Leu Glu Asp Gln 1115 1120 1125 Lys Glu Gly Arg Ser Thr Asn Lys Glu Asn Pro Ser Lys Ala Leu 1130 1135 1140 Ile Glu Arg Pro Ser Gln Asn Asn Ile Gly Ile Gln Thr Met Glu 1145 1150 1155 Cys Ser Leu Arg Val Pro Glu Thr Val Ser Ala Ala Thr Gln Thr 1160 1165 1170 Ile Lys Asn Val Cys Glu Gln Gly Thr Ser Thr Val Asp Gln Asn 1175 1180 1185 Phe Gly Lys Gln Asp Ala Thr Val Gln Thr Glu Arg Gly Ser Gly 1190 1195 1200 Glu Lys Pro Val Ser Ala Pro Gly Asp Asp Thr Glu Ser Leu His 1205 1210 1215 Ser Gln Gly Glu Glu Glu Phe Asp Met Pro Gln Pro Pro His Gly 1220 1225 1230 His Val Leu His Arg His Met Arg Thr Ile Arg Glu Val Arg Thr 1235 1240 1245 Leu Val Thr Arg Val Ile Thr Asp Val Tyr Tyr Val Asp Gly Thr 1250 1255 1260 Glu Val Glu Arg Lys Val Thr Glu Glu Thr Glu Glu Pro Ile Val 1265 1270 1275 Glu Cys Gln Glu Cys Glu Thr Glu Val Ser Pro Ser Gln Thr Gly 1280 1285 1290 Gly Ser Ser Gly Asp Leu Gly Asp Ile Ser Ser Phe Ser Ser Lys 1295 1300 1305 Ala Ser Ser Leu His Arg Thr Ser Ser Gly Thr Ser Leu Ser Ala 1310 1315 1320 Met His Ser Ser Gly Ser Ser Gly Lys Gly Ala Gly Pro Leu Arg 1325 1330 1335 Gly Lys Thr Ser Gly Thr Glu Pro Ala Asp Phe Ala Leu Pro Ser 1340 1345 1350 Ser Arg Gly Gly Pro Gly Lys Leu Ser Pro Arg Lys Gly Val Ser 1355 1360 1365 Gln Thr Gly Thr Pro Val Cys Glu Glu Asp Gly Asp Ala Gly Leu 1370 1375 1380 Gly Ile Arg Gln Gly Gly Lys Ala Pro Val Thr Pro Arg Gly Arg 1385 1390 1395 Gly Arg Arg Gly Arg Pro Pro Ser Arg Thr Thr Gly Thr Arg Glu 1400 1405 1410 Thr Ala Val Pro Gly Pro Leu Gly Ile Glu Asp Ile Ser Pro Asn 1415 1420 1425 Leu Ser Pro Asp Asp Lys Ser Phe Ser Arg Val Val Pro Arg Val 1430 1435 1440 Pro Asp Ser Thr Arg Arg Thr Asp Val Gly Ala Gly Ala Leu Arg 1445 1450 1455 Arg Ser Asp Ser Pro Glu Ile Pro Phe Gln Ala Ala Ala Gly Pro 1460 1465 1470 Ser Asp Gly Leu Asp Ala Ser Ser Pro Gly Asn Ser Phe Val Gly 1475 1480 1485 Leu Arg Val Val Ala Lys Trp Ser Ser Asn Gly Tyr Phe Tyr Ser 1490 1495 1500 Gly Lys Ile Thr Arg Asp Val Gly Ala Gly Lys Tyr Lys Leu Leu 1505 1510 1515 Phe Asp Asp Gly Tyr Glu Cys Asp Val Leu Gly Lys Asp Ile Leu 1520 1525 1530 Leu Cys Asp Pro Ile Pro Leu Asp Thr Glu Val Thr Ala Leu Ser 1535 1540 1545 Glu Asp Glu Tyr Phe Ser Ala Gly Val Val Lys Gly His Arg Lys 1550 1555 1560 Glu Ser Gly Glu Leu Tyr Tyr Ser Ile Glu Lys Glu Gly Gln Arg 1565 1570 1575 Lys Trp Tyr Lys Arg Met Ala Val Ile Leu Ser Leu Glu Gln Gly 1580 1585 1590 Asn Arg Leu Arg Glu Gln Tyr Gly Leu Gly Pro Tyr Glu Ala Val 1595 1600 1605 Thr Pro Leu Thr Lys Ala Ala Asp Ile Ser Leu Asp Asn Leu Val 1610 1615 1620 Glu Gly Lys Arg Lys Arg Arg Ser Asn Val Ser Ser Pro Ala Thr 1625 1630 1635 Pro Thr Ala Ser Ser Ser Ser Ser Thr Thr Pro Thr Arg Lys Ile 1640 1645 1650 Thr Glu Ser Pro Arg Ala Ser Met Gly Val Leu Ser Gly Lys Arg 1655 1660 1665 Lys Leu Ile Thr Ser Glu Glu Glu Arg Ser Pro Ala Lys Arg Gly 1670 1675 1680 Arg Lys Ser Ala Thr Val Lys Pro Gly Ala Val Gly Ala Gly Glu 1685 1690 1695 Phe Val Ser Pro Cys Glu Ser Gly Asp Asn Thr Gly Glu Pro Ser 1700 1705 1710 Ala Leu Glu Glu Gln Arg Gly Pro Leu Pro Leu Asn Lys Thr Leu 1715 1720 1725 Phe Leu Gly Tyr Ala Phe Leu Leu Thr Met Ala Thr Thr Ser Asp 1730 1735 1740 Lys Leu Ala Ser Arg Ser Lys Leu Pro Asp Gly Pro Thr Gly Ser 1745 1750 1755 Ser Glu Glu Glu Glu Glu Phe Leu Glu Ile Pro Pro Phe Asn Lys 1760 1765 1770 Gln Tyr Thr Glu Ser Gln Leu Arg Ala Gly Ala Gly Tyr Ile Leu 1775 1780 1785 Glu Asp Phe Asn Glu Ala Gln Cys Asn Thr Ala Tyr Gln Cys Leu 1790 1795 1800 Leu Ile Ala Asp Gln His Cys Arg Thr Arg Lys Tyr Phe Leu Cys 1805 1810 1815 Leu Ala Ser Gly Ile Pro Cys Val Ser His Val Trp Val His Asp 1820 1825 1830 Ser Cys His Ala Asn Gln Leu Gln Asn Tyr Arg Asn Tyr Leu Leu 1835 1840 1845 Pro Ala Gly Tyr Ser Leu Glu Glu Gln Arg Ile Leu Asp Trp Gln 1850 1855 1860 Pro Arg Glu Asn Pro Phe Gln Asn Leu Lys Val Leu Leu Val Ser 1865 1870 1875 Asp Gln Gln Gln Asn Phe Leu Glu Leu Trp Ser Glu Ile Leu Met 1880 1885 1890 Thr Gly Gly Ala Ala Ser Val Lys Gln His His Ser Ser Ala His 1895 1900 1905 Asn Lys Asp Ile Ala Leu Gly Val Phe Asp Val Val Val Thr Asp 1910 1915 1920 Pro Ser Cys Pro Ala Ser Val Leu Lys Cys Ala Glu Ala Leu Gln 1925 1930 1935 Leu Pro Val Val Ser Gln Glu Trp Val Ile Gln Cys Leu Ile Val 1940 1945 1950 Gly Glu Arg Ile Gly Phe Lys Gln His Pro Lys Tyr Lys His Asp 1955 1960 1965 Tyr Val Ser His 1970 

What is claimed is:
 1. A method for detecting DNA damage in a tissue sample, comprising the steps of: contacting a biological sample with a ligand which binds to human 53Bp1, said ligand associated with a label which provides a detectable signal; examining said sample for the presence of signal concentrated in foci of 53Bp1 in said sample, wherein the presence of concentrated foci is indicative of DNA damage and the presence of diffuse signal is indicative of a normal sample.
 2. The method according to claim 1, further comprising immobilizing said sample prior to said contacting or examining steps.
 3. The method according to claim 1 wherein said biological sample is a white blood cell.
 4. The method according to claim 1 wherein said biological sample is a biopsy specimen.
 5. The method according to claim 3 or 4, wherein said sample is selected from the group consisting of peripheral blood, saliva, urine, a cell scraping, exudate, a buccal sample, sputum, and cervical scraping.
 6. The method according to claim 1 wherein said ligand is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or a recombinant antibody of classes IgG, IgM, IgA, IgD and IgE; a Fab, Fab′ or F(ab′)2, or Fc antibody fragment thereof, a single chain Fv antibody fragment, a recombinant construct comprising a complementarity determining region of an antibody directed to 53Bp1, a synthetic antibody or chimeric antibody or humanized antibody construct which shares sufficient CDRs of an antibody to 53Bp1 to retain functionally equivalent binding characteristics of an antibody that binds 53Bp1.
 7. The method according to claim 1, wherein said examining step comprises performing immunofluorescent microscopy or immunohistochemical analysis with a suitable ligand.
 8. The method according to claim 1, wherein said 53Bp1 comprises a fragment comprising amino acid residues 1220 to 1711 of SEQ ID NO:
 2. 9. A diagnostic reagent comprising a ligand that binds to human 53Bp1, said ligand associated with a detectable label.
 10. The reagent according to claim 8, wherein said ligand is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or a recombinant antibody of classes IgG, IgM, IgA, IgD and IgE; a Fab, Fab′ or F(ab′)2, or Fc antibody fragment thereof, a single chain Fv antibody fragment, a recombinant construct comprising a complementarity determining region of an antibody directed to 53Bp1, a synthetic antibody or chimeric antibody or humanized antibody construct that shares sufficient CDRs of an antibody to 53Bp1 to retain functionally equivalent binding characteristics of an antibody that binds 53Bp1.
 11. The reagent according to claim 10, which is an anti53Bp1 monoclonal antibody.
 12. The reagent according to claim 9, wherein said label is a fluorescent label, a colloidal gold label or an enzymatic label.
 13. The reagent according to claim 9, wherein said 53Bp1 comprises a fragment comprising amino acid residues 1220 to 1711 of SEQ ID NO:
 2. 14. A diagnostic kit for detecting DNA damage in a biological sample, said kit comprising a diagnostic reagent which is a ligand which binds to human 53Bp1, said ligand associated with a detectable label, and suitable components for detection of said label.
 15. The kit according to claim 1, wherein said 53Bp1 comprises a fragment comprising amino acid residues 1220 to 1711 of SEQ ID NO:
 2. 16. A composition that antagonizes or inhibits the biological activity or expression of 53Bp1.
 17. The composition according to claim 16, comprising a ligand of 53Bp1 selected from the group consisting of a polyclonal antibody, a monoclonal antibody or a recombinant antibody of classes IgG, IBM, IgA, IgD and IgE; a Fab, Fab′ or F(ab′)2, or Fc antibody fragment thereof, a single chain Fv antibody fragment, a recombinant construct comprising a complementarity determining region of an antibody directed to 53Bp1, a synthetic antibody or chimeric antibody or humanized antibody construct which shares sufficient CDRs of an antibody to 53Bp1 to retain functionally equivalent binding characteristics of an antibody that binds 53Bp1.
 18. The composition according to claim 16, wherein said composition is a synthetic chemical compound.
 19. The composition according to claim 16, wherein said composition is a peptidomimetic of 53Bp1.
 20. The composition according to claim 16, wherein said composition is an anti-sense oligonucleotide that inhibits expression of said 53Bp1, and thereby inhibits the biological activity thereof.
 21. A method of screening test compounds to identify a composition that inhibits or antagonizes the biological activity of 53Bp1, said method comprising employing a 53Bp1 ligand associated with a detectable label to detect the expression of 53Bp1 in a cell contacted with a test compound or to detect the presence or number of 53Bp1 induced nuclear foci in cells contacted with a test compound.
 22. The method according to claim 21, further comprising the steps of: (a) providing a first cell which is contacted with a suitable amount of a test compound; (b) providing a control comprising a cell identical to said first cell but without exposure to said test compound; (c) exposing said cells (a) and (b) to a DNA damaging agent; (d) contacting said cells (c) with a ligand that binds 53Bp1, said ligand associated with a detectable label; (e) examining said cells (d) for detection of the signal generated by said label, which signal indicates the presence and number of 53Bp1 nuclear foci, and (f) comparing the signals generated by the labels in said two cells, wherein a lesser detectable signal in said cell (a) compared with the detectable signal in said cell (b) indicates that said test compound has inhibited the presence and/or number of 53Bp1 foci in cell (a) and is a 53Bp1 inhibitor.
 23. The method according to claim 21, wherein said method steps are selected from the group consisting of cellular assays, enzymatic assays, assays for the detection of DNA synthesis and competition assays.
 24. The method according to claim 21, wherein said 53Bp1 comprises a fragment comprising amino acid residues 1220 to 1711 of SEQ ID NO:
 2. 25. A 53Bp1 inhibitor identified by the method of claim
 21. 26. A method of retarding the growth of a cancer cell, said method comprising administering to the site of a cancer cell a 53Bp1 inhibitor or ligand.
 27. The method according to claim 26, wherein said administering step comprises administering said inhibitor to a cell ex vivo.
 28. The method according to claim 26, wherein said administering step comprises administering said inhibitor to a mammal having a cancer in vivo.
 29. A method of targeting a tumor cell for delivery of a therapeutic agent, comprising administering to a patient bearing a tumor containing 53Bp1 foci a ligand that binds to 53Bp1, said ligand associated with a compound that retards the growth of, or kills, said tumor cell.
 30. The method according to claim 29, wherein said compound is selected from the group consisting of a radionucleotide, a toxin, a bi-specific antibody and an anticancer drug optionally linked to a protein or peptide.
 31. The method according to claim 29, wherein said ligand binds a fragment of 53Bp1 comprising amino acid residues 1220 to 1711 of SEQ ID NO:
 2. 